Methods and compositions based on protein interactions with mastermind

ABSTRACT

The invention is directed to methods of modulating Notch signal transduction and to complexes of the protein Mastermind with proteins identified as interacting with Mastermind by a two-hybrid screen as well as a complex of Mastermind (Mam) with Mip1, or a complex of Mam with Mip30, or a complex of Mam with Mip6. Methods of screening the complexes for efficacy in treating and/or preventing certain diseases and disorders, particularly hyperproliferative and cancerous conditions are also provided. The invention includes nucleic acid and amino acid sequences of Mip30 or Mip6, as well as fragments and derivatives thereof.

1. FIELD OF THE INVENTION

The present invention is directed to modulating signal transduction.

2. BACKGROUND OF THE INVENTION

2.1 Notch Signal Transduction

Genetic and molecular studies have led to the identification of a groupof genes which define distinct elements of the Notch signaling pathway.While the identification of these various elements has come exclusivelyfrom Drosophila using genetic tools as the initial guide, subsequentanalyses have lead to the identification of homologous proteins invertebrate species including humans. See, generally, Artavanis-Tsakonaset al., 1995, Science 268:225-232.

The Drosophila Notch gene encodes an ˜300 kD transmembrane protein thatacts as a receptor in a cell-cell signaling mechanism controlling cellfate decisions throughout development (reviewed, e.g., inArtavanis-Tsakonas et al., 1995, Science 268:225-232). Closely relatedhomologs of Drosophila Notch have been isolated from a number ofvertebrate species, including humans, with multiple paralogsrepresenting the single Drosophila gene in vertebrate genomes. Theisolation of cDNA clones encoding the C-terminus of a human Notchparalog, originally termed h N, has been reported (Stifani et al., 1992,Nature Genetics 2:119-127). The encoded protein is designated humanNotch2 because of its close relationship to the Notch2 proteins found inother species (Weinmaster et al., 1992, Development 116:931-941). Thehallmark Notch2 structures are common to all the Notch-related proteins,including, in the extracellular domain, a stretch of 34 to 36 tandemEpidermal Growth Factor-like (EGF) repeats (fewer EGF repeats in Notch 3and 4) and three Lin-12/Notch repeats (LN repeats), and, in theintracellular domain, 6 Ankyrin repeats and a PEST-containing region.Like Drosophila Notch and the related C. elegans genes lin-12 and glp-1(Sternberg, 1993, Current Biology 3:763-765; Greenwald, 1994, CurrentOpinion in Genetics and Development 4:556-562), the vertebrate Notchhomologs play a role in a variety of developmental processes bycontrolling cell fate decisions (reviewed, e.g., in Blaumueller andArtavanis-Tsakonas, 1997, Persp. on Dev. Neurobiol. 4:325-343). Forfurther human Notch sequences, see International Publication WO 92/19734and WO 99/04746.

The extracellular domain of Notch generally carries 36 Epidermal GrowthFactor-like (EGF) repeats, two of which (repeats 11 and 12) have beenimplicated in interactions with the Notch ligands Serrate and Delta.Delta and Serrate are membrane bound ligands with EGF homologousextracellular domains, which interact physically with Notch on adjacentcells to trigger signaling.

Functional analyses involving the expression of truncated forms of theNotch receptor have indicated that receptor activation depends on thesix cdc10/ankyrin repeats in the intracellular domain. Deltex andSuppressor of Hairless, whose over-expression results in an apparentactivation of the pathway, associate with those repeats.

Deltex is a cytoplasmic protein which contains a ring zinc finger.Suppressor of Hairless on the other hand, is the Drosophila homolog ofCBF1, a mammalian DNA binding protein involved in the Epstein-Barrvirus-induced immortalization of B cells. It has been demonstrated that,at least in cultured cells, Suppressor of Hairless associates with thecdc10/ankyrin repeats in the cytoplasm and translocates into the nucleusupon the interaction of the Notch receptor with its ligand Delta onadjacent cells (Fortini and Artavanis, 1994, Cell 79:273-282). Theassociation of Hairless, a novel nuclear protein, with Suppressor ofHairless has been documented using the yeast two hybrid system;therefore, it is believed that the involvement of Suppressor of Hairlessin transcription is modulated by Hairless (Brou et al., 1994, Genes Dev.8:2491; Knust et al. 1992, Genetics 129:803).

Finally, it is known that Notch signaling results in the activation ofat least certain basic helix-loop-helix (bHLH) genes within the Enhancerof Split complex (Delidakis et al., 1991, Genetics 129:803).

The generality of the Notch pathway manifests itself at differentlevels. At the genetic level, many mutations exist which affect thedevelopment of a very broad spectrum of cell types in Drosophila.Knockout mutations in mice are embryonic lethals consistent with afundamental role for Notch function (Swiatek et al., 1994, Genes Dev.8:707). Mutations in the Notch pathway in the hematopoietic system inhumans are associated with lymphoblastic leukemia (Ellison et al., 1991,Cell 66:649-661). Finally the expression of mutant forms of Notch indeveloping Xenopus embryos interferes profoundly with normal development(Coffman et al., 1993, Cell 73:659). Increased level of Notch expressionis found in some malignant tissue in humans (International PublicationWO 94/07474).

The expression patterns of Notch in the Drosophila embryo are complexand dynamic. The Notch protein is broadly expressed in the early embryo,and subsequently becomes restricted to uncommitted or proliferativegroups of cells as development proceeds. In the adult, expressionpersists in the regenerating tissues of the ovaries and testes (reviewedin Fortini et al., 1993, Cell 75:1245-1247; Jan et al., 1993, Proc.Natl. Acad. Sci. USA 90:8305-8307; Sternberg, 1993, Curr. Biol.3:763-765; Greenwald, 1994, Curr. Opin. Genet. Dev. 4:556-562;Artavanis-Tsakonas et al., 1995, Science 268:225-232). Studies of theexpression of Notch1, one of three known vertebrate homologs of Notch,in zebrafish and Xenopus, have shown that the general patterns aresimilar; with Notch expression associated in general with non-terminallydifferentiated, proliferative cell populations. Tissues with highexpression levels include the developing brain, eye and neural tube(Coffman et al., 1990, Science 249:1438-1441; Bierkamp et al., 1993,Mech. Dev. 43:87-100). While studies in mammals have shown theexpression of the corresponding Notch homologs to begin later indevelopment, the proteins are expressed in dynamic patterns in tissuesundergoing cell fate determination or rapid proliferation (Weinmaster etal., 1991, Development 113:199-205; Reaume et al., 1992, Dev. Biol.154:377-387; Stifani et al., 1992, Nature Genet. 2:119-127; Weinmasteret al., 1992, Development 116:931-941; Kopan et al., 1993, J. Cell Biol.121:631-641; Lardelli et al., 1993, Exp. Cell Res. 204:364-372; Lardelliet al., 1994, Mech. Dev. 46:123-136; Henrique et al., 1995, Nature375:787-790; Horvitz et al., 1991, Nature 351:535-541; Franco del Amo etal., 1992, Development 115:737-744). Among the tissues in whichmammalian Notch homologs are first expressed are the pre-somiticmesoderm and the developing neuroepithelium of the embryo. In thepre-somitic mesoderm, expression of Notch1 is seen in all of themigrated mesoderm, and a particularly dense band is seen at the anterioredge of pre-somitic mesoderm. This expression has been shown to decreaseonce the somites have formed, indicating a role for Notch in thedifferentiation of somatic precursor cells (Reaume et al., 1992, Dev.Biol. 154:377-387; Horvitz et al., 1991, Nature 351:535-541). Similarexpression patterns are seen for mouse Delta (Simske et al., 1995,Nature 375:142-145).

Within the developing mammalian nervous system, expression patterns ofNotch homologs have been shown to be prominent in particular regions ofthe ventricular zone of the spinal cord, as well as in components of theperipheral nervous system, in an overlapping but non-identical pattern.Notch expression in the nervous system appears to be limited to regionsof cellular proliferation, and is absent from nearby populations ofrecently differentiated cells (Weinmaster et al., 1991, Development113:199-205; Reaume et al., 1992, Dev. Biol. 154:377-387; Weinmaster etal., 1992, Development 116:931-941; Kopan et al., 1993, J. Cell Biol.121:631-641; Lardelli et al., 1993, Exp. Cell Res. 204:364-372; Lardelliet al., 1994, Mech. Dev. 46:123-136; Henrique et al., 1995, Nature375:787-790; Horvitz et al., 1991, Nature 351:535-541). A rat Notchligand is also expressed within the developing spinal cord, in distinctbands of the ventricular zone that overlap with the expression domainsof the Notch genes. The spatio-temporal expression pattern of thisligand correlates well with the patterns of cells committing to spinalcord neuronal fates, which demonstrates the usefulness of Notch as amarker of populations of cells for neuronal fates (Henrique et al.,1995, Nature 375:787-790). This has also been suggested for vertebrateDelta homologues, whose expression domains also overlap with those ofNotch1 (Larsson et al., 1994, Genomics 24:253-258; Fortini et al., 1993,Nature 365:555-557; Simske et al., 1995, Nature 375:142-145). In thecases of the Xenopus and chicken homologues, Delta is actually expressedonly in scattered cells within the Notch1 expression domain, as would beexpected from the lateral specification model, and these patterns“foreshadow” future patterns of neuronal differentiation (Larsson etal., 1994, Genomics 24:253-258; Fortini et al., 1993, Nature365:555-557).

Other vertebrate studies of particular interest have focused on theexpression of Notch homologs in developing sensory structures, includingthe retina, hair follicles and tooth buds. In the case of the Xenopusretina, Notch1 is expressed in the undifferentiated cells of the centralmarginal zone and central retina (Coffman et al., 1990, Science249:1439-1441; Mango et al., 1991, Nature 352:811-815). Studies in therat have also demonstrated an association of Notch1 with differentiatingcells in the developing retina have been interpreted to suggest thatNotch1 plays a role in successive cell fate choices in this tissue(Lyman et al., 1993, Proc. Natl. Acad. Sci. USA 90:10395-10399).

A detailed analysis of mouse Notch1 expression in the regeneratingmatrix cells of hair follicles was undertaken to examine the potentialparticipation of Notch proteins in epithelial/mesenchymal inductiveinteractions (Franco del Amo et al., 1992, Development 115:737-744).Such a role had originally been suggested for Notch1 based on the itsexpression in rat whiskers and tooth buds (Weinmaster et al., 1991,Development 113:199-205). Notch1 expression was instead found to belimited to subsets of non-mitotic, differentiating cells that are notsubject to epithelial/mesenchymal interactions, a finding that isconsistent with Notch expression elsewhere.

Expression studies of Notch proteins in human tissue and cell lines havealso been reported. The aberrant expression of a truncated Notch1 RNA inhuman T-cell leukemia results from a translocation with a breakpoint inNotch1 (Ellisen et al., 1991, Cell 66:649-661). A study of human Notch1expression during hematopoiesis has suggested a role for Notch1 in theearly differentiation of T-cell precursors (Mango et al., 1994,Development 120:2305-2315). Additional studies of human Notch1 andNotch2 expression have been performed on adult tissue sections includingboth normal and neoplastic cervical and colon tissue. Notch1 and Notch2appear to be expressed in overlapping patterns in differentiatingpopulations of cells within squamous epithelia of normal tissues thathave been examined and are clearly not expressed in normal columnarepithelia, except in some of the precursor cells. Both proteins areexpressed in neoplasias, in cases ranging from relatively benignsquamous metaplasias to cancerous invasive adenocarcinomas in whichcolumnar epithelia are replaced by these tumors (Mello et al., 1994,Cell 77:95-106).

Insight into the developmental role and the general nature of Notchsignaling has emerged from studies with truncated, constitutivelyactivated forms of Notch in several species. These recombinantlyengineered Notch forms, which lack extracellular ligand-binding domains,resemble the naturally occurring oncogenic variants of mammalian Notchproteins and are constitutively activated using phenotypic criteria(Greenwald, 1994, Curr. Opin. Genet. Dev. 4:556; Fortini et al., 1993,Nature 365:555-557; Coffman et al., 1993, Cell 73:659-671; Struhl etal., 1993, Cell 69:1073; Rebay et al., 1993, Genes Dev. 7:1949; Kopan etal., 1994, Development 120:2385; Roehl et al., 1993, Nature 364:632).

-   -   Ubiquitous expression of activated Notch in the Drosophila        embryo suppresses neuroblast segregation without impairing        epidermal differentiation (Struhl et al., 1993, Cell 69:331;        Rebay et al., 1993, Genes Dev. 7:1949).    -   Persistent expression of activated Notch in developing imaginal        epithelia likewise results in an overproduction of epidermis at        the expense of neural structures (Struhl et al., 1993, Cell        69:331).    -   Neuroblast segregation occurs in temporal waves that are delayed        but not prevented by transient expression of activated Notch in        the embryo (Struhl et al., 1993, Cell 69:331).    -   Transient expression in well-defined cells of the Drosophila eye        imaginal disc causes the cells to ignore their normal inductive        cues and to adopt alternative cell fates (Fortini et al., 1993,        Nature 365:555-557).    -   Studies utilizing transient expression of activated Notch in        either the Drosophila embryo or the eye disc indicate that once        Notch signaling activity has subsided, cells may recover and        differentiate properly or respond to later developmental cues        (Fortini et al., 1993, Nature 365:555-557; Struhl et al., 1993,        Cell 69:331).

For a general review on the Notch pathway and Notch signaling, seeArtavanis-Tsakonas et al., 1995, Science 268:225-232.

Ligands, cytoplasmic effectors and nuclear elements of Notch signalinghave been identified in Drosophila, and vertebrate counterparts havealso been cloned (reviewed in Artavanis-Tsakonas et al., 1995, Science268:225-232). While protein interactions between the various elementshave been documented, the biochemical nature of Notch signaling remainselusive. Expression of truncated forms of Notch reveal that Notchproteins without transmembrane and extracellular domains aretranslocated to the nucleus both in transgenic flies and in transfectedmammalian or Drosophila cells (Lieber et al., 1993, Genes andDevelopment 7:1949-1965; Fortini et al., 1993, Nature 365:555-557; Ahmadet al., 1995, Mechanisms of Development 53:78-85; Zagouras et al., 1995,Proc. Natl. Acad. Sci. USA 92:6414-6418). Sequence comparisons betweenmammalian and Drosophila Notch molecules, along with deletion analysis,have found two nuclear localization sequences that reside on either sideof the ankyrin repeats (Stifani et al., 1992, Nature Genetics 2:119-127;Lieber et al., 1993, Genes and Development 7:1949-1965; Kopan et al.,1994, Development 120:2385-2396). These findings prompted thespeculation that Notch may be directly participating in nuclear eventsby means of a proteolytic cleavage and subsequent translocation of theintracellular fragment into the nucleus. However, conclusive functionalevidence for such a hypothesis remains elusive (Artavanis-Tsakonas etal., 1995, Science 268:225-232).

2.2 Mam

Mastermind encodes a novel ubiquitous nuclear protein involved in theNotch pathway as shown by genetic analysis (Smoller et al., 1990, GenesDev. 4:1688). Two human homologs of Mastermind have been cloned, MAML1and MAML2 (Wu et al., 2000, Nature Genetics 26:484-489; see FIGS. 1-6).Mastermind contains an amino-terminal basic domain and two acid domains,one of which is in the carboxy terminus, and has been shown to localizeto nuclear bodies. FIG. 5 is a schematic of the Mastermind domains andtheir location. Drosophila Mastermind is 1596 amino acids in length andhas an unusually large number of homopolymer repeats (glutamine, glycineand asparagine) that are separated by regions of charged amino acids, anarrangement similar to nuclear regulatory proteins. Mastermind has beenshown to bind to the ankyrin repeat domain of all four known mammalianNotch proteins and its expression has been shown to amplifyNotch-induced transcription, and thus, Mastermind functions as atranscriptional co-activator for Notch signal transduction (Wu et al.,2000, Nature Genetics 26:484-489).

2.3 SUMO Conjugation

SUMO (small ubiquitin-related modifier) is the best characterized memberof a growing family of ubiquitin-related proteins. It resemblesubiquitin in its structure, its ability to be ligated to other proteins,as well as in the mechanism of ligation. However, in contrast toubiquitinization, often the first step on a one-way road to proteindegradation, sumolation does not seem to mark proteins for degradation.In fact, sumolation may even function as an antagonist of ubiquitin inthe degradation of selected proteins. The SUMO conjugation machinery isevolutionarily conserved and has been described in organisms rangingfrom yeast to man. SUMO first undergoes an ATP-dependent activation by aheterodimeric complex (Uba2p/Aos1p) and is conjugated to Aos1p(activating enzyme) through a thioester bond. The SUMO protein is thentransferred, through another thioester bond, to a SUMO-conjugatingenzyme, Ubc9. Additional components of the SUMO conjugation pathway havenot been identified, and it is likely that SUMO is conjugated to aprotein substrate through direct transfer from Ubc9. The types ofproteins known to date that are modified by SUMO participate in a widespectrum of nuclear processes, including nuclear transport, kinetochoreand centromere function, recombination, transcription and nuclear bodystructure. Consequently, any protein or signaling pathway that caninfluence SUMO conjugation could have a profound effect on nuclearfunctions. For a general review of the SUMO conjugation pathway, seeMelchior, 2000, Ann. Rev. Cell Dev. Biol. 16:591-526.

2.4 Mip1/Uba2p

Drosophila Uba2p (Mip1) is one of two subunits that comprise theactivating enzyme for SUMO. Homologs of the Uba2p gene have been clonedfrom several species, including humans. See FIG. 8 for an amino acidcomparison of different homologs of Uba2p.

Citation or identification of any reference in Section 2 or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery ofinteractions of Mastermind (Mam) with the Mip1, Mip30 and Mip6 proteins,as well as the isolation of Mip30 and Mip6 nucleic and amino acidsequences. The present invention is also based, in part, on the novelobservation that an increase in Notch signal transduction results in anincrease in sumolation in a cell, thus demonstrating the interdependenceof the Notch signal transduction pathway and SUMO conjugation.

Mastermind is a member of the Notch family of proteins and is involvedin the regulation of cell fate and differentiation through Notchsignaling. As described in Section 2.2, supra, Mastermind binds to theankyrin repeat domain of Notch. Mastermind also binds Mip1, Mip30 andMip6. Mip1, also called Uba2p, which, as discussed in Sections 2.3 and2.4, supra, is part of the SUMO conjugation machinery, in particular,one of two subunits that comprise the SUMO activating enzyme. Sumolationof cellular proteins has been shown to alter their subcellularlocalization and result in longer half-lives, i.e., stabilization of theproteins. Mutations resulting in aberrant sumolation, e.g., disruptionof the gene encoding SUMO, leads to severe growth defects in yeast andphenotypes such as aberrant mitosis, increase in telomere length, anddefects in chromosomal segregation. It is well known that the centrosomeis involved in mitosis and fidelity of chromosome segregation and thatmalfunctioning centrosomes can lead to missegregation of the chromosomesduring mitosis, which appears to be involved in tumorigenesis, i.e.cancer formation. See, e.g., Doxsey, 1998, Nat. Genet. 20:104-106. Thus,the compositions and methods of the present invention are useful instudying cell fate and differentiation and tumorigenesis, and instudying telomere regulation and chromosome segregation and foridentifying modulators of cell fate and differentiation andtumorigenesis, and in identifying modulators of telomere regulation andchromosome segregation.

The present invention is directed to methods of identifying a moleculethat alters Notch signal transduction in a cell comprising contactingthe cell with one or more candidate molecules; and measuring the amountof sumolation in the cell, wherein an increase or decrease in the amountof sumolation relative to said amount in a cell not so contacted withone or more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction. The present invention is alsodirected to methods of identifying a molecule that alters Notch signaltransduction in a cell comprising recombinantly expressing within thecell one or more candidate molecules; and measuring the amount ofsumolation in the cell, wherein an increase or decrease in the amount ofsumolation relative to said amount in a cell not so contacted with oneor more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction. The present invention is alsodirected to methods of identifying a molecule that alters Notch signaltransduction in a cell comprising microinjecting into the cell one ormore candidate molecules; and measuring the amount of sumolation in thecell, wherein an increase or decrease in the amount of sumolationrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules alterNotch signal transduction.

Sumolation, or SUMO conjugation activity, can be measured, e.g., by anincrease or decrease in the conjugation of SUMO to target proteins. Thetotal cellular complement of protein targets or specific protein targetscan be analyzed. The SUMO protein can be introduced as a transgene ineither an epitope-tagged form or an un-tagged form. Alternatively, theextent of endogenous SUMO conjugation activity can be assessed, e.g.,using anti-SUMO antibodies, or by Western blot analysis in which theresults would be amenable to quantification by densitometry. Further,since SUMO conjugation of a protein often influences the intracellularlocalization of the protein, an assay based upon the localization of aspecific target protein can be used. Also, since SUMO conjugation of aprotein often stabilizes the protein since SUMO competes with the sametarget lysine as ubiquitin, sumolation can be measured by measuring thestability, i.e., half-life, of the target protein, e.g., by Western blotanalysis.

The present invention is directed to methods of identifying a moleculethat alters sumolation activity in a cell comprising contacting the cellwith one or more candidate molecules; and measuring the amount of Notchsignal transduction in the cell, wherein an increase or decrease in theamount of Notch signal transduction relative to said amount in a cellnot so contacted with one or more of the candidate molecules indicatesthat the candidate molecules alter sumolation activity. Another methodof identifying a molecule that alters sumolation in a cell comprisesrecombinantly expressing within the cell one or more candidatemolecules; and measuring the amount of Notch signal transduction in thecell, wherein an increase or decrease in the amount of Notch signaltransduction relative to said amount in a cell not so contacted with oneor more of the candidate molecules indicates that the candidatemolecules alter sumolation activity. Yet another method of identifying amolecule that alters sumolation activity in a cell comprisesmicroinjecting into the cell one or more candidate molecules; andmeasuring the amount of Notch signal transduction in the cell, whereinan increase or decrease in the amount of Notch signal transductionrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules altersumolation activity.

Notch signal transduction or Notch function can be measured using assayscommonly known in the art, e.g., by the ability of Notch to activatetranscription of a gene in the Enhancer of split complex, e.g., mγ, mδ,m5; or to activate transcription of vestigial, cut, or the HES1 gene. Anin vitro transcription assay utilizing HES1 has been described (Wu etal., 2000, Nature Genetics 26:484-489; Jarriault et al., 1995, Nature377:355-358). Thus, increased levels of mγ, mδ, m5, vestigial, cut orHES1 mRNA or protein indicates an increased level of Notch signaltransduction or Notch function. Conversely, decreased levels of mγ, mδ,m5, vestigial, cut or HES1 mRNA or protein indicates a decreased levelof Notch signal transduction or Notch function. Further, activation ofNotch signal transduction results in the inhibition of differentiationof precursor cells. See, U.S. Pat. No. 5,780,300. Thus, Notch signaltransduction can also be measured by assaying for differentiation ofprecursor cells. Maintenance of the differentiation state of theprecursor cell indicates active Notch signal transduction. A change inthe differentiation state of the precursor cell indicates inactive Notchsignal transduction. Additionally, reporter constructs with a reportergene under the control of a promoter containing a Notch-responsivepromoter element can also be used to detect Notch signal transduction.For example, the EBNA2 response element from the TP-1 promoter can beused in such a reporter construct.

The present invention is also directed to methods of inhibiting Notchsignal transduction in a cell comprising contacting the cell with anantagonist of sumolation in an amount sufficient to inhibit Notch signaltransduction. Further, the present invention is directed to methods ofagonizing Notch signal transduction in a cell comprising contacting thecell with an agonist of sumolation in an amount sufficient to agonizeNotch signal transduction. The present invention is also directed tomethods of inhibiting sumolation activity in a cell comprisingcontacting the cell with an antagonist of Notch signal transduction inan amount sufficient to inhibit sumolation activity, as well as, methodsof agonizing sumolation activity in a cell comprising contacting thecell with an agonist of Notch signal transduction in an amountsufficient to agonize sumolation activity. Agonists and antagonists ofboth sumolation and Notch signal transduction are well known in the art,and can also be identified using the methods of the present invention,infra.

The present invention is directed to certain compositions comprising andmethods for production of protein complexes of Mam with a protein thatinteracts with (i.e., binds to) Mam. As used herein, “Mam-IP” refers toa Mam-interacting protein, e.g. Mip1, Mip30, Mip6. Specifically, theinvention is directed to complexes of Mam, and derivatives, fragmentsand analogs of Mam, with Mip1, Mip30 or Mip6, and their derivatives,fragments and analogs (a complex of Mam and Mip1 or Mam and Mip30 or Mamand Mip6 is designated as Mam:Mip1 or Mam:Mip30 or Mam:Mip6,respectively, herein). The present invention is further directed tomethods of screening for proteins that interact with Mam and/or Mip1,Mip30, or Mip6, or with derivatives, fragments or analogs of Mam and/orMip1, Mip30 or Mip6.

The present invention is also directed to Mip30 and Mip6 proteins,fragments and derivatives, and their encoding nucleic acids, as well asantibodies to the proteins, fragments and derivatives of Mip30 and Mip6.

Methods for production of the Mam:Mip1, Mam:Mip30 and Mam:Mip6complexes, and derivatives and analogs of the complexes and/orindividual proteins, e.g., by recombinant means, are also provided.Pharmaceutical compositions are also provided.

The invention is further directed to methods for modulating (i.e.,inhibiting or enhancing) the activity of a Mam:Mip1, Mam:Mip30 orMam:Mip6 complex, and/or Mip30 or Mip6. The protein components of aMam:Mip1, Mam:Mip30 and Mam:Mip6 complexes have been implicated inphysiological processes including, but not limited to, disease anddisorders of cell fate and differentiation and aberrant mitotic events,such as defects in chromosome segregation. Accordingly, the presentinvention is directed to methods for screening Mam:Mip1, Mam:Mip30 orMam:Mip6 complexes or Mip30 or Mip6, as well as derivatives and analogsof the complexes or Mip30 or Mip6, for the ability to alter a cellfunction, particularly a cell function in which Mam, Mip1, Mip30 and/orMip6 has been implicated, as non-exclusively listed, supra.

The present invention is also directed to therapeutic and prophylactic,as well as diagnostic, prognostic, and screening methods andcompositions based upon the Mam:Mip1, Mam:Mip30 or Mam:Mip6 complexes(and the nucleic acids encoding the individual proteins that participatein the complex). Therapeutic compounds of the invention include, but arenot limited to, Mam:Mip1, Mam:Mip30 or Mam:Mip6 complexes, and a complexwhere one or both members of the complex is a derivative, fragment,homolog or analog of Mam, Mip1, Mip30 or Mip6; antibodies to and nucleicacids encoding the foregoing; and antisense nucleic acids to thenucleotide sequences encoding the complex components. Diagnostic,prognostic and screening kits are also provided.

Animal models and methods of screening for modulators (i.e., agonists,and antagonists) of the activity of Mam:Mip1, Mam:Mip30 or Mam:Mip6complexes and/or the individual proteins are also provided.

Methods of identifying molecules that inhibit, or alternatively, thatincrease formation of a Mam:Mip1, Mam:Mip30 or Mam:Mip6 complex are alsoprovided.

The methods of the present invention can be carried out either in vitroor in vivo.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the nucleotide (SEQ ID NO:1) and amino acid (SEQ IDNO:2) sequences of Drosophila Mastermind (GenBank Accession No. X54251).

FIG. 2 sets forth the nucleotide (SEQ ID NO:3) and amino acid (SEQ IDNO:4) sequences of a human homolog of Mastermind, MAML1 (GenBankAccession No. NM_(—)014757).

FIG. 3 sets forth the nucleotide (SEQ ID NO:5) and amino acid (SEQ IDNO:6) sequences of another human homolog of Mastermind, MAML2 (GenBankAccession No. AB058719).

FIG. 4 is a comparison between the amino acid sequence of DrosophilaMastermind (SEQ ID NO:2) and two human homologs of Mastermind, MAML1(SEQ ID NO:4) and MAML2 (SEQ ID NO:6), and also sets forth a consensussequence (SEQ ID NO:7) based on the sequence comparison.

FIG. 5 is a schematic diagram showing the basic and two acidic domainsof Mastermind as well as the regions of Mastermind that are responsiblefor binding to Notch and to Mip1, Mip30 and Mip6, and the regionsresponsible for transcriptional activation and for inducing sumolation.

FIG. 6 sets forth the nucleotide (SEQ ID NO:8) and the amino acidsequences (SEQ ID NO:9) of Mip1 (Uba2p).

FIG. 7 is a comparison between amino acid sequence of Neurospora(T51083) (SEQ ID NO:10), S. pombe (T39623) (SEQ ID NO:11), S. cerevisiae(UNK_(—)68186217) (SEQ ID NO:12), human (UNK_(—)68168211) (SEQ IDNO:13), mouse (UNK_(—)681862122) (SEQ ID NO:14), Drosophila(AF193553_(—)1:) (SEQ ID NO:9), C. elegans (UNK_(—)68186214) (SEQ IDNO:15) and Arabadopsis (AC06841_(—)24:) (SEQ ID NO:16) homologs of Mip1(Uba2p). A consensus sequence is also generated (SEQ ID NO21).

FIG. 8 is a chart setting forth the amino acid length of each Mip1protein compared in FIG. 7, as well as the amino acid location of theUBACT repeat domain and UBA/THIF family domain for each homolog.

FIG. 9 is a schematic of the Mip1 protein showing the location of theUBA/THIF-type NAD/FAD family domain (amino acids 12-155), the UBACTrepeat domain (amino acids 359-506), the bipartite nuclear localizationsignal (NLS) (amino acids 154-171), and Mastermind interacting domain(amino acids 458-700).

FIG. 10 sets forth the nucleotide (SEQ ID NO:17) and the amino acidsequences (SEQ ID NO:18) of Mip30.

FIG. 11 is a schematic of the Mip30 protein showing the location of themotifs present in Mip30. Three prominent motifs were identified,C2H2-type zinc fingers (amino acids 28-51, 71-97, 104-127, 341-364,383-407, 414-437 and 482-504), an A+T hook domain (amino acids 164-176)and a bipartite nuclear localization signal (NLS) (amino acids 301-318).

FIG. 12 sets forth the nucleotide (SEQ ID NO:19) and the amino acidsequences (SEQ ID NO:20) of Mip6. The minimal Mam interacting domain ofMip6 known is amino acids 374-625.

FIG. 13 is a graph showing Mam-Mip complex driven transcription in atwo-hybrid analysis in yeast. The yeast strain EGY48 was co-transformedwith a plasmid encoding a Mam-Gal4 DNA binding domain fusion protein andeither a plasmid encoding Mip1, Mip30 or Mip6 fused to the E. coli B42transactivation domain, or a control plasmid (pJG4-5). The DNA bindingdomain was derived from plasmid pEG202. A Mam-Mip interaction isdemonstrated by activation of transcription from a lacZ transgene andreported in terms of arbitrary β-galactosidase units. In the presence ofglucose, the expression of Mam is repressed and only a very low level ofβ-galactosidase activity is detected. In the presence of galactose, theexpression of Mam is induced and a large increase in β-galactosidaseactivity is observed with those proteins that interact with Mam.Extracts were prepared and activity measured from equal number of cells.

FIG. 14 shows that Mastermind is localized to subnuclear domains byindirect immunofluorescence analysis in 293T cells. Drosophila Mam wastagged at its amino terminus with the Flag epitope (Ciaccia and Pierce,1992, IBI Flag Epitope 1:4-5) and expressed from the pcDNA3 vector(Invitrogen, Carlsbad, Calif.) in a human kidney epithelial cell line(293T). Mam was visualized using the anti-Flag monoclonal antibody M2(Sigma, St. Louis, Mo.) and a Cy-2-conjugated goat anti-mouse IgGsecondary antibody obtained from Jackson ImmunoResearch Laboratories,Inc., West Grove, Pa. Nuclei were counterstained with DAPI.

FIG. 15 shows that Mastermind localizes to nuclear bodies in 293T cell,as determined by co-localization with the PML oncogene product, thesignature protein for nuclear bodies. Hemagglutinin (HA) epitope-taggedMam was visualized with a rabbit polyclonal anti-HA antibody (obtainedfrom Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) and aCy-5-conjugated goat anti-rabbit IgG secondary antibody (obtained fromJackson ImmunoResearch Laboratories, West Grove, Pa.). PML wasvisualized with the PG-M3 monoclonal antibody (obtained from Santa CruzBiotechnology, Inc., Santa Cruz, Calif.) and a Cy-2-conjugated goatanti-mouse secondary antibody (obtained from Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.).

FIG. 16 demonstrates that Mastermind induces Notch relocalization in293T cells by indirect immunofluorescence. Mastermind and the entireintracellular domain of Drosophila Notch were co-expressed from pcDNA3vectors (obtained from Invitrogen, Carlsbad, Calif.). IntracellularNotch was visualized with the 9C6 monoclonal antibody (Rebay, 1983,Thesis, Yale University) and a Cy-2-conjugated goat anti-mouse IgGsecondary antibody. Flag epitope-tagged Mam was visualized with ananti-Flag polyclonal antibody and a Cy-5-conjugated goat anti-rabbitsecondary antibody. Nuclei were counterstained with DAPI. In the absenceof Mam, intracellular Notch is homogeneously distributed throughout thenucleoplasm; however, when co-expressed with Mam, intracellular Notchaccumulates in nuclear bodies.

FIG. 17 shows that Mastermind induces Mip1 localization to nuclearbodies. Mip1 was tagged at its amino terminus with the HA epitope andexpressed from the pcDNA3 vector. Mip1 was visualized with a rabbitpolyclonal anti-HA antibody and a Cy-2-conjugated goat anti-rabbit IgGsecondary antibody. Mam was visualized with the M2 anti-Flag monoclonalantibody and a Cy-2-conjugated goat anti-mouse secondary antibody.Nuclei were counterstained with DAPI. In the absence of Mam, Mip1appears to be homogeneously distributed throughout the nucleoplasm. Whenco-expressed with Mam, Mip1 accumulates in nuclear bodies.

FIG. 18 is a western blot of total cell lysates prepared fromtransfected 293T cell showing that Mastermind is an activator ofsumolation. Cells were co-transfected with an equal amount of plasmid(pcDNA3) encoding HA-tagged Drosophila SUMO protein and increasingamounts of a plasmid (pcDNA3) encoding Flag-tagged Mam. Expression ofMam increases the level of SUMO conjugation to cellular proteins.SUMO-conjugated proteins were detected with a monoclonal anti-HAantibody (obtained from BabCO, Richmod, Calif.), an HRP-conjugated goatanti-mouse IgG secondary antibody (obtained from Santa CruzBiotechnology, Santa Cruz, Calif.) and the Super Signal DuraWestchemoluminescence detection system (Pierce, Rockford, Ill.). Blockingand antibody incubations were carried out in 1×PBS; 0.25% Tween 20; 5%non-fat dry milk (Carnation); 5% goat serum (Sigma, St. Louis, Mo.,catalog # G-6767). Blots were incubated with primary and secondaryantibodies for 1 hour each. Detection was performed as permanufacturer's (Pierce's) instructions.

FIGS. 19A and 19B are western blots of total cell lysates prepared from293T cells and show that Mastermind is a general activator of SUMOconjugation activity in that Mam increases the conjugation of SUMO-1,SUMO-2 and SUMO-3 to cellular proteins. In FIG. 19A, the cells weretransfected with equal amounts of a plasmid encoding HA epitope-taggedSUMO and a control plasmid (lane 1) or a plasmid encoding HAepitope-tagged SUMO and a plasmid encoding Flag epitope-tagged Mam (lane2). In FIG. 19B, the cells were transfected with equal amounts of aplasmid encoding HA-tagged SUMO-2 and a control plasmid (lane 3), equalamounts of a plasmid encoding HA-epitope-tagged SUMO-3 and a controlplasmid (lane 4), equal amounts of a plasmid encoding HA-epitope-taggedSUMO-2 and a plasmid encoding Flag epitope-tagged Mam (lane 5), andequal amounts of a plasmid encoding HA-epitope-tagged SUMO-3 and aplasmid encoding Flag epitope-tagged Mam (lane 6). Western blots wereprobed and developed as described for FIG. 16.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, upon the identification ofproteins that interact with Mastermind, a protein involved in the Notchsignal transduction pathway. The interacting proteins Mip1, Mip30 andMip6 were found to form a complex under physiological conditions withMam. The Mam:Mip1, Mam:Mip30 and Mam:Mip6 complexes, by virtue of theinteraction, are implicated in modulating the functional activities ofMam and its binding partners, in particular, Mip1, Mip30 and Mip6. Suchfunctional activities include physiological processes including, but notlimited to, disorders of cell fate and differentiation and disorders todue aberrant chromosome segregation. The present invention is alsodirected to novel nucleic and amino acid sequences of Mip30 and Mip6 andmethods and compositions relating thereto.

The present invention is directed to methods of identifying a moleculethat alters Notch signal transduction in a cell comprising contactingthe cell with one or more candidate molecules; and measuring the amountof sumolation in the cell, wherein an increase or decrease in the amountof sumolation relative to said amount in a cell not so contacted withone or more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction. The present invention is alsodirected to methods of identifying a molecule that alters Notch signaltransduction in a cell comprising recombinantly expressing within thecell one or more candidate molecules; and measuring the amount ofsumolation in the cell, wherein an increase or decrease in the amount ofsumolation relative to said amount in a cell not so contacted with oneor more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction. The present invention is alsodirected to methods of identifying a molecule that alters Notch signaltransduction in a cell comprising microinjecting into the cell one ormore candidate molecules; and measuring the amount of sumolation in thecell, wherein an increase or decrease in the amount of sumolationrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules alterNotch signal transduction.

Sumolation, or SUMO conjugation activity, can be measured, e.g., by anincrease or decrease in the conjugation of SUMO to target proteins. Thetotal cellular complement of protein targets or specific protein targetscan be analyzed. The SUMO protein can be introduced as a transgene ineither an epitope-tagged form or an un-tagged form. Alternatively, theextent of endogenous SUMO conjugation activity can be assessed, e.g.,using anti-SUMO antibodies, or by Western blot analysis in which theresults would be amenable to quantification by densitometry. Further,since SUMO conjugation of a protein often influences the intracellularlocalization of the protein, an assay based upon the localization of aspecific target protein can be used. Also, since SUMO conjugation of aprotein often stabilizes the protein since SUMO competes with the sametarget lysine as ubiquitin, sumolation can be measured by measuring thestability, i.e., half-life, of the target protein, e.g., by Western blotanalysis.

The present invention is directed to methods of identifying a moleculethat alters sumolation activity in a cell comprising contacting the cellwith one or more candidate molecules; and measuring the amount of Notchsignal transduction in the cell, wherein an increase or decrease in theamount of Notch signal transduction relative to said amount in a cellnot so contacted with one or more of the candidate molecules indicatesthat the candidate molecules alter sumolation activity. Another methodof identifying a molecule that alters sumolation in a cell comprisesrecombinantly expressing within the cell one or more candidatemolecules; and measuring the amount of Notch signal transduction in thecell, wherein an increase or decrease in the amount of Notch signaltransduction relative to said amount in a cell not so contacted with oneor more of the candidate molecules indicates that the candidatemolecules alter sumolation activity. Yet another method of identifying amolecule that alters sumolation activity in a cell comprisesmicroinjecting into the cell one or more candidate molecules; andmeasuring the amount of Notch signal transduction in the cell, whereinan increase or decrease in the amount of Notch signal transductionrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules altersumolation activity.

Notch signal transduction or Notch function can be measured using assayscommonly known in the art, e.g., by the ability of Notch to activatetranscription of a gene in the Enhancer of split complex, e.g., mγ, mδ,m5; or to activate transcription of vestigial, cut, or the HES1 gene. Anin vitro transcription assay utilizing HES1 has been described (Wu etal., 2000, Nature Genetics 26:484-489; Jarriault et al., 1995, Nature377:355-358). Thus, increased levels of mγ, mδ, m5, vestigial, cut orHES1 mRNA or protein indicates an increased level of Notch signaltransduction or Notch function. Conversely, decreased levels of mγ, mδ,m5, vestigial, cut or HES1 mRNA or protein indicates a decreased levelof Notch signal transduction or Notch function. Further, activation ofNotch signal transduction results in the inhibition of differentiationof precursor cells. See, U.S. Pat. No. 5,780,300. Thus, Notch signaltransduction can also be measured by assaying for differentiation ofprecursor cells. Maintenance of the differentiation state of theprecursor cell indicates active Notch signal transduction. A change inthe differentiation state of the precursor cell indicates inactive Notchsignal transduction. See U.S. Pat. Nos. 5,780,300 and 6,083,904 formethods of measuring the differentiation state of a cell and changesthereof based on Notch signal transduction. Additionally, reporterconstructs with a reporter gene under the control of a promotercontaining a Notch-responsive promoter element can also be used todetect Notch signal transduction. For example, the EBNA2 responseelement from the TP-1 promoter can be used in such a reporter construct.

The present invention is also directed to methods of inhibiting Notchsignal transduction in a cell comprising contacting the cell with anantagonist of sumolation in an amount sufficient to inhibit Notch signaltransduction. Further, the present invention is directed to methods ofagonizing Notch signal transduction in a cell comprising contacting thecell with an agonist of sumolation in an amount sufficient to agonizeNotch signal transduction. The present invention is also directed tomethods of inhibiting sumolation activity in a cell comprisingcontacting the cell with an antagonist of Notch signal transduction inan amount sufficient to inhibit sumolation activity, as well as, methodsof agonizing sumolation activity in a cell comprising contacting thecell with an agonist of Notch signal transduction in an amountsufficient to agonize sumolation activity. Agonists and antagonists ofboth sumolation and Notch signal transduction are well known in the art,and can also be identified using the methods of the present invention,infra. For example, an antagonist of sumolation is a dominant negativeform a Mip1, or other protein in the sumolation conjugation pathway. Anillustrative example of a dominant negative form of Mip1 is a form thatcontains a mutated ADP binding domain such that ADP does not bind.Agonists of Notch include, but are not limited to dominant active formsof Notch, including the intracellular domain of Notch, Delta andSerrate. An illustrative dominant negative form of Notch is a form whichlacks the intracellular domain. See International Publications WO00/02897, WO 97/01571, WO96/27610 and WO 97/18822 for illustrativeexamples of Notch signal transduction pathway agonists and antagonists.Other antagonists of both Notch signal transduction and SUMO conjugationare antibodies which are specific for the members of the pathway, e.g.,anti-Notch, anti-Mip 1, anti-Ubc9. Other antagonists include antisensenucleic acids which bind to and block translation of mRNAs encodingmembers of the pathway.

The present invention is directed to methods of screening for proteinsthat interact with (e.g., bind to) Mastermind (Mam). The inventionfurther relates to Mam complexes, in particular Mam complexes with oneof the following proteins: Mip1, Mip30 or Mip6. The invention furtherrelates to complexes of derivatives, analogs and fragments of Mam, withMip1, Mip30 or Mip6 or derivatives, analogs and fragments thereof ofthese Mam interacting proteins (“Mam-IPs”). In a preferred embodimentsuch complexes bind an anti-Mam:Mam-IP complex antibody. In a specificembodiment, complexes of human Mam with a human Mam-IP protein areprovided.

The invention also provides methods of producing and/or isolatingMam:Mam-IP complexes. In a specific embodiment, the invention providesmethods of using recombinant DNA techniques to express Mam and itsbinding partner (or fragments, derivatives or homologs of one or bothmembers of the complex) either where both binding partners are under thecontrol of one heterologous promoter (i.e., a promoter not naturallyassociated with the native gene encoding the particular complexcomponent) or where each is under the control of a separate heterologouspromoter.

The present invention also provides the nucleotide sequence of Mip30 andMip-6, and their encoded amino acid sequences. The invention furtherrelates to a Mip30 or Mip6 protein, derivatives (including but notlimited to fragments) and homologs and analogs thereof, as well as tonucleic acids encoding the Mip30 or Mip6 protein, derivatives, fragmentsand homologs. The invention further provides for a Mip30 or Mip6 proteinand gene encoding the protein, from many different species, particularlyvertebrates, and more particularly mammals. In a preferred embodiment,the Mip30 or Mip6 protein and gene is of human origin. Production of theforegoing proteins and derivatives, e.g., by recombinant methods, isalso provided in the present invention.

The present invention further relates to a Mip30 or Mip6 derivative oranalog that is functionally active, i.e., capable of displaying one ormore known functional activities associated with a full length(wild-type) Mip30 or Mip6. Such functional activities include, but arenot limited to, the ability to form a complex with Mam, antigenicity[ability to bind (or compete with Mip30 or Mip6 for binding) to ananti-Mip30 or anti-Mip6 antibody, respectively], immunogenicity (abilityto generate an antibody that binds to Mip30 or Mip6, respectively), etc.

Methods of diagnosis, prognosis, and screening for diseases anddisorders associated with aberrant levels of a Mam:Mam-IP complex, oraberrant levels of a Mip30 or Mip6 protein, are provided. The inventionalso provides methods of treating or preventing diseases or disordersassociated with aberrant levels of a Mam:Mam-IP complex or with aberrantlevels of a Mip30 or Mip6 protein, or aberrant levels of activity of oneor more of the components of the complex, comprising administration ofthe Mam:Mam-IP complex, or administration of the Mip30 or Mip6 protein,or administration of modulators of Mam:Mam-IP complex formation oractivity (e.g., antibodies that bind the Mam:Mam-IP complex, ornon-complexed Mam or its binding partner or a fragmentthereof—preferably the fragment containing the portion of Mam or theMam-IP that is directly involved in complex formation) The methods alsoinclude administering mutants of Mam or the Mam-IP that increase ordecrease binding affinity, administering small moleculeinhibitors/enhancers of complex formation, or administering antibodiesthat either stabilize or neutralize the complex, etc.

Methods of assaying a Mam:Mam-IP complex, or of assaying a Mip30 or Mip6protein, for activity as therapeutics or diagnostics as well as methodsof screening for Mam:Mam-IP complex, Mip30 or Mip6 modulators (i.e.,inhibitors, agonists and antagonists) are also provided.

The methods of the present invention can be performed either in vitro orin vivo.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

5.1 Mam:Mam-IP Complexes and Mip30 and Mip6 Proteins, Derivatives andAnalogs

The present invention provides Mam:Mam-IP complexes, and in particularaspects, complexes of Mam and Mip1, Mam and Mip30 and Mam:Mip6. In apreferred embodiment, the Mam:Mam-IP complex is a complex of humanproteins.

The invention also relates to complexes of derivatives (includingfragments) and analogs of Mam with a Mam-IP, complexes of Mam withderivatives (including fragments) and analogs of a Mam-IP, and complexesof derivatives (including fragments) and analogs of Mam and derivatives(including fragments) and analogs of a Mam-IP. As used herein, fragment,derivative or analog of a Mam:Mam-IP complex includes a complex whereinone or both members of the complex is a fragment(s), derivative(s) oranalog(s) of the wild-type Mam or Mam-IP protein. Preferably, theMam:Mam-IP complex in which one or both members of the complex is afragment, derivative or analog of the wild type protein is afunctionally active Mam:Mam-IP complex. In particular aspects, thenative proteins, derivatives or analogs of Mam and/or the Mam-IP arefrom animals, e.g., mouse, rat, pig, cow, dog, monkey, human, fly, frog.In another aspect the native proteins, derivatives or analogs of Mamand/or the Mam-IP are from plants.

Accordingly, the present invention provides methods of screeningMam:Mam-IP complexes, particularly complexes of Mam with Mip1, Mip30 andMip6 proteins, as well as derivatives and analogs of the Mam:Mam-IPcomplexes, and methods of screening Mip30 and Mip6 proteins for theability to alter cell functions, particularly those cell functions inwhich Mam and/or a Mam-IP has been implicated. Such functions include,but not limited to, physiological processes such as signal transduction,post-translational protein modification, and pathological processes suchas degenerative disorders including neurodegenerative disease,hyperproliferative disorders including tumorigenesis and tumorprogression.

Other functions of the complexes, aside from the ability to altercellular function, include binding to an anti-Mam:Mam-IP complexantibody, as well as other activities as described in the art. Forexample, derivatives or analogs of the Mam:Mam-IP complex that have thedesired immunogenicity or antigenicity can be used in immunoassays, forimmunization, for inhibition of Mam:Mam-IP complex activity, etc.Derivatives or analogs of the Mam:Mam-IP complex that retain or enhance,or alternatively lack or inhibit, a property of interest, e.g.,participation in a Mam:Mam-IP complex, can be used as inducers, orinhibitors, respectively, of such a property and its physiologicalcorrelates. A specific embodiment relates to a Mam:Mam-IP complex of afragment of a Mam protein and/or a fragment of a Mam-IP protein that canbe bound by an anti-Mam and/or anti-Mam-IP antibody or by an antibodyspecific for a Mam:Mam-IP complex, when such fragment is included in aMam:Mam-IP complex.

Fragments and other derivatives or analogs of Mam:Mam-IP complexes canbe tested for the desired activity by procedures known in the art,including but not limited to the assays described in Section 5.7, infra.

The invention further relates to Mip30 or Mip6 protein as well asderivatives and homologs and analogs of Mip30 or Mip6 protein. In oneembodiment a human Mip30 or Mip6 gene and protein is provided. Inspecific aspects, the native protein, fragment, derivative or analog ofMip30 or Mip6 protein is from animals, e.g., mouse, rat, pig, cow, dog,monkey, human, fly, or frog. In another aspect, the native protein,fragment, derivative or analog of Mip30 or Mip6 protein is from plants.In other specific embodiments, the fragment, derivative or analog isfunctionally active, i.e., capable of exhibiting one or more functionalactivity associated with wild type Mip30 or Mip6 protein, e.g., abilityto bind Mam, immunogenicity or antigenicity.

The nucleotide sequences encoding Mam and Mip1 from several species,including humans, are known and are provided in FIGS. 1-4 and 6-7,respectively. Nucleic acids encoding Mam, Mip1, Mip30 or Mip6 can beobtained by any method known in the art, e.g, by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequenceand/or by cloning from a cDNA or genomic library using anoligonucleotide specific for the gene sequence, e.g., as described inSection 5.2, infra. Due to the degeneracy of the genetic code, the term“Mam, Mip1, Mip30 or Mip6 gene”, as used herein, refers not only to thenaturally occurring nucleotide sequence but also encompasses all theother degenerate DNA sequences that encode a Mam, Mip1, Mip30 or Mip6polypeptide, respectively. Computer programs, such as Entrez, can beused to browse the database, and retrieve any amino acid sequence andgenetic sequence data of interest by accession number. These databasescan also be searched to identify sequences with various degrees ofsimilarities to a query sequence using programs, such as FASTA andBLAST, which rank the similar sequences by alignment scores andstatistics. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Altschul et al., 1997, supra). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used (seehttp://www.ncbi.nlm.nih.gov).

Homologs, e.g., of nucleic acids encoding Mam, Mip1, Mip30 or Mip6 ofspecies other than human, or other related sequences, e.g., paralogs,can be obtained by low, moderate or high stringency hybridization withall or a portion of the particular sequence as a probe using methodswell known in the art for nucleic acid hybridization and cloning, e.g.,as described in Section 5.2, infra, for Mip30, or Mip6 nucleotidesequences.

The Mam, Mip1, Mip30 or Mip6 proteins as depicted in FIGS. 1-12, (SEQ IDNOS:2, 4, 6, 7 (Mam); SEQ ID NOS:9, 10, 11, 12, 13, 14, 15, 16 (Mip1);SEQ ID NO:18 (Mip30); and SEQ ID NO:20 (Mip6)) either alone or in acomplex, can be obtained by methods well known in the art for proteinpurification and recombinant protein expression. For recombinantexpression of one or more of the proteins, the nucleic acid containingall or a portion of the nucleotide sequence encoding the protein can beinserted into an appropriate expression vector, i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted protein coding sequence. The necessary transcriptional andtranslational signals can also be supplied by the native promoter forMam or any Mam-IP genes, and/or their flanking regions.

A variety of host-vector systems may be utilized to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors or bacteriatransformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

In a preferred embodiment, a Mam:Mam-IP complex is obtained byexpressing the entire Mam sequence and a Mam-IP coding sequence in thesame cell, either under the control of the same promoter or under twoseparate promoters. In yet another embodiment, a derivative, fragment orhomolog of Mam and/or a derivative, fragment or homolog of a Mam-IP arerecombinantly expressed. Preferably the derivative, fragment or homologof Mam and/or of the Mam-IP protein forms a complex with a bindingpartner identified by a binding assay, such as the modified yeast twohybrid system described in Section 5.8.1 infra, and more preferablyforms a complex that binds to an anti-Mam:Mam-IP complex antibody.

Any of the methods described in Section 5.2, infra, for the insertion ofDNA fragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleotide sequences encoding Mam and a Mam-IP (e.g.,Mip1, Mip30, Mip6, or a derivative, fragment or homolog thereof), may beregulated by a second nucleotide sequence so that the gene or genefragment thereof is expressed in a host transformed with the recombinantDNA molecule(s). For example, expression of the proteins may becontrolled by any promoter/enhancer known in the art. In a specificembodiment, the promoter is not native to the gene for Mam or forMam-IP.

Promoters which may be used include but are not limited to the SV40early promoter (Bernoist and Chambon, 1981, Nature 290:304-310); thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto et al., 1980, Cell 22:787-797); the Herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445);the regulatory sequences of the metallothionein gene (Brinster et al.,1982, Nature 296:39-42); prokaryotic expression vectors such as theβ-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad.Sci. USA 75:3727-3731) or the tac promoter (DeBoer et al., 1983, Proc.Natl. Acad. Sci. USA 80:21-25, see also Useful Proteins from RecombinantBacteria: in Scientific American 1980, 242:79-94); plant expressionvectors comprising the nopaline synthetase promoter (Herrar-Estrella etal., 1984, Nature 303:209-213) or the cauliflower mosaic virus 35S RNApromoter (Garder et al., 1981, Nucleic Acids Res. 9:2871), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter; and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been utilizedin transgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald1987, Hepatology 7:425-515), insulin gene control region which is activein pancreatic beta cells (Hanahan et al., 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adams et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell Biol. 7:1436-1444), mousemammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinckert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha-1antitrypsin gene control region which is active in liver (Kelsey et al.,1987, Genes and Devel. 1: 161-171), beta globin gene control regionwhich is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells of thebrain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani 1985,Nature 314:283-286), and gonadotrophic releasing hormone gene controlregion which is active in gonadotrophs of the hypothalamus (Mason etal., 1986, Science 234:1372-1378).

In a specific embodiment, a vector is used that comprises a promoteroperably linked to nucleotide sequences encoding Mam and/or a Mam-IP(e.g., Mip1, Mip30, Mip6), or a fragment, derivative or homolog thereof,one or more origins of replication, and optionally, one or moreselectable markers (e.g., an antibiotic resistance gene). In a preferredembodiment, a vector is used that comprises a promoter operably linkedto nucleotide sequences encoding both Mam and a Mam-IP, one or moreorigins of replication, and optionally, one or more selectable markers.

In another specific embodiment, an expression vector containing thecoding sequence, or a portion thereof, of Mam and a Mam-IP eithertogether or separately, is made by subcloning the gene sequences intothe EcoRI restriction site of one of the three pGEX vectors (glutathioneS-transferase expression vectors; Smith and Johnson, 1988, Gene 7:3140;Promega Corp., Madison, Wis.). This allows for the expression ofproducts in the correct reading frame.

Expression vectors containing the sequences of interest can beidentified by three general approaches: (a) nucleic acid hybridization,(b) presence or absence of marker gene function, and (c) expression ofthe inserted sequences. In the first approach, Mam, Mip1, Mip30 or Mip6,or other Mam-IP sequences can be detected by nucleic acid hybridizationto probes comprising sequences homologous and complementary to theinserted sequences. In the second approach, the recombinant vector/hostsystem can be identified and selected based upon the presence or absenceof certain marker functions (e.g., binding to an anti-Mam, anti-Mam-IP,or anti-Mam:Mam-IP complex antibody, resistance to antibiotics,occlusion body formation in baculovirus, etc.) caused by insertion ofthe sequences of interest in the vector. For example, if a Mam or Mam-IPgene, or portion thereof, is inserted within the marker gene sequence ofthe vector, recombinants containing the Mam or Mam-IP fragment will beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingfor Mam, Mip1, Mip30 or Mip6 products expressed by the recombinantvector. Such assays can be based, for example, on the physical orfunctional properties of the interacting species in in vitro assaysystems, e.g., formation of a Mam:Mam-IP complex or immunoreactivity toantibodies specific for the protein.

Once recombinant Mam, Mip1, Mip30 or Mip6 molecules are identified andthe complexes or individual proteins are isolated, several methods knownin the art can be used to propagate them. Once a suitable host systemand growth conditions have been established, recombinant expressionvectors can be propagated and amplified in quantity. As previouslydescribed, the expression vectors or derivatives which can be usedinclude, but are not limited to, human or animal viruses such asvaccinia virus or adenovirus; insect viruses such as baculovirus; yeastvectors; bacteriophage vectors such as lambda phage; and plasmid andcosmid vectors.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequence, or modifies or processes theexpressed protein in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically-engineered Mam and/or Mam-IP may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation, etc.)of proteins. Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign protein isachieved. For example, expression in a bacterial system can be used toproduce an unglycosylated core protein, while expression in mammaliancells can ensure native glycosylation of a heterologous mammalianprotein. Furthermore, different vector/host expression systems mayeffect processing reactions to different extents.

In other specific embodiments, the Mam and/or Mam-IP, or fragment,homolog or derivative thereof, may be expressed as a fusion or chimericprotein product comprising the protein, fragment, homolog, or derivativejoined via a peptide bond to a heterologous protein sequence of adifferent protein. Such chimeric products can be made by ligating theappropriate nucleic acids encoding the desired amino acids to each otherin the proper coding frame by methods known in the art, and expressingthe chimeric products in a suitable host by methods commonly known inthe art. Alternatively, such a chimeric product can be made by proteinsynthetic techniques, e.g., by use of a peptide synthesizer. Chimericgenes comprising portions of Mam and/or Mip1, Mip30, or Mip6, fused toany heterologous protein-encoding sequences may be constructed. Aspecific embodiment relates to a chimeric protein comprising a fragmentof Mam and/or a Mam-IP, or a fragment of Mip1, Mip30, or Mip6 protein,of at least six amino acids.

In a specific embodiment, fusion proteins are provided that contain theinteracting domains of the Mam protein and a Mam-IP (e.g., Mip1, Mip30and Mip6) and/or, optionally, a hetero-functional reagent, such as apeptide linker between the two domains, where such a reagent promotesthe interaction of Mam and Mam-IP binding domains. These fusion proteinsmay be particularly useful where the stability of the interaction isdesirable (due to the formation of the complex as an intra-molecularreaction), for example in production of antibodies specific to theMam:Mam-IP complex.

In particular, Mam and/or Mip1, Mip30 or Mip6 derivatives can be made byaltering their respective sequence by substitutions, additions ordeletions that provide for functionally equivalent molecules. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences thatencode substantially the same amino acid sequence as a Mam or Mam-IPgene can be used in the practice of the present invention. These includebut are not limited, to a nucleotide sequence comprising all or aportion of Mam, Mip1, Mip30, or Mip6 gene that is altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change.

Likewise, Mam and Mam-IP derivatives of the invention include, but arenot limited to, those containing, as a primary amino acid sequence, allor part of the amino acid sequence of Mam or a Mam-IP, including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of Mam or Mam-IP consisting of at least 6(continuous) amino acids of Mam or a Mam-IP are provided. In otherembodiments, the fragment consists of at least about 10, 20, 30, 40, 50,60, 70, 80, or 90 amino acids of Mam or a Mam-IP. In specificembodiments, such fragments are not larger than about 35, 50, 75, 100,125, 150, 175, 200, 300, 400 or 500 amino acids. Derivatives or analogsof Mam and Mam-IPs, include, but are not limited to, moleculescomprising regions that are substantially homologous to Mam or Mam-IPs,in various embodiments, by at least about 30%, 40%, 50%, 60%, 70%, 80%,90% or 95% identity over an amino acid sequence of identical size orwhen compared to an aligned sequence in which the alignment is done by acomputer homology program known in the art, or whose encoding nucleicacid is capable of hybridizing to the complement (e.g., the inversecomplement) of a sequence encoding Mam or a Mam-IP under stringent,moderately stringent, or nonstringent conditions, as described infra.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87-2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.,1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Altschul et al., 1997,supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used (see http://www.ncbi.nlm.nih.gov). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

The Mam, Mip1, Mip30 and Mip6 derivatives and analogs of the inventioncan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or protein level.For example, the cloned Mam or Mam-IP gene sequence can be modified byany of numerous strategies known in the art (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The sequences can becleaved at appropriate sites with restriction endonuclease(s), followedby further enzymatic modification if desired, isolated, and ligated invitro. In the production of a gene encoding a derivative or analog ofMam or a Mam-IP, care should be taken to ensure that the modified generetains the original translational reading frame, uninterrupted bytranslational stop signals.

Additionally, the Mam and/or Mam-IP-encoding nucleotide sequence can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypre-existing ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, chemical mutagenesis and in vitro site-directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551-6558), useof TAB™ linkers (Pharmacia), etc.

Once a recombinant cell expressing Mam and/or a Mam-IP protein, orfragment or derivative thereof, is identified, the individual geneproduct or complex can be isolated and analyzed. This is achieved byassays based on the physical and/or functional properties of the proteinor complex, including, but not limited to, radioactive labeling of theproduct followed by analysis by gel electrophoresis, immunoassay,cross-linking to marker-labeled product, etc.

The Mam:Mam complex, or Mam, Mip1, Mip30 or Mip6 protein, can beisolated and purified by standard methods known in the art (either fromnatural sources or recombinant host cells expressing the complexes orproteins), including but not restricted to column chromatography (e.g.,ion exchange, affinity, gel exclusion, reversed-phase high pressure,fast protein liquid, etc.), differential centrifugation, differentialsolubility, or by any other standard technique used for the purificationof proteins. Functional properties may be evaluated using any suitableassay known in the art.

Alternatively, once a Mam-IP or its derivative is identified, the aminoacid sequence of the protein can be deduced from the nucleotide sequenceof the chimeric gene from which it was encoded. As a result, the proteinor its derivative can be synthesized by standard chemical methods knownin the art (see, e.g., Hunkapiller et al., 1984, Nature 310: 105-111).

In a specific embodiment of the present invention, such Mam:Mam-IPcomplex, or Mam, Mip1, Mip30 or Mip6 protein, whether produced byrecombinant DNA techniques, chemical synthesis methods, or bypurification from native sources, include but are not limited to thosecontaining as a primary amino acid sequence all or part of the aminoacid sequences substantially as depicted in FIGS. 1-12 (SEQ ID NOS:2, 4,6, 7 (Mam); SEQ ID NOS:9, 10, 11, 12, 13, 14, 15, 16 (Mip1); SEQ IDNO:18 (Mip30); and SEQ ID NO:20 (Mip6)), as well as fragments and otheranalogs and derivatives thereof, including proteins homologous thereto.

Manipulations of Mam and/or Mam-IP sequences may be made at the proteinlevel. Included within the scope of the invention are derivatives ofcomplexes of Mam and/or Mam-IP fragments, derivatives or analogs thereofthat are differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, prenylation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, etc.Any of numerous chemical modifications may be carried out by knowntechniques, including but not limited to specific chemical cleavage bycyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄,acetylation, formylation, oxidation, reduction, metabolic synthesis inthe presence of tunicamycin, etc.

In specific embodiments, the Mam and/or Mam-IP sequences are modified toinclude a fluorescent label. In another specific embodiment, the Mamand/or the Mam-IP are modified to have a heterofunctional reagent; suchheterofunctional reagents can be used to crosslink the protein to othermembers of the complex or to other Mam-IPs.

In addition, analogs and derivatives of Mam and/or a Mam-IP, or analogsand derivatives of Mam, Mip1, Mip30 or Mip6 protein, can be chemicallysynthesized. For example, a peptide corresponding to a portion of Mamand/or a Mam-IP, which comprises the desired domain or mediates thedesired activity in vitro (e.g., Mam:Mam-IP complex formation) can besynthesized by use of a peptide synthesizer. Furthermore, if desired,non-classical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the Mam and/or a Mam-IP.Non-classical amino acids include but are not limited to the D-isomersof the common amino acids, amino isobutyric acid, 4-aminobutyric acid(4-Abu), 2-aminobutyric acid (2-Abu), 6-amino hexanoic acid (e-Ahx),2-amino isobutyric acid (Aib), 3-amino propionoic acid, ornithine,norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteicacid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, C-methyl amino acids, N-methyl amino acids, and amino acidanalogs in general. Furthermore, classical or non-classical amino acidscan be D (dextrorotary) or L (levorotary).

In cases where natural products are suspected of being mutant or areisolated from new species, the amino acid sequence of Mam, or a Mam-IPisolated from the natural source, as well as those expressed in vitro,or from synthesized expression vectors in vivo or in vitro, can bedetermined from analysis of the DNA sequence, or alternatively, bydirect sequencing of the isolated protein. Such analysis may beperformed by manual sequencing or through use of an automated amino acidsequenator.

The Mam:Mam-IP complex, or Mam, Mip1, Mip30 or Mip6 protein, may also beanalyzed by hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl.Acad. Sci. USA 78:3824-3828). A hydrophilicity profile can be used toidentify the hydrophobic and hydrophilic regions of the proteins, andhelp predict their orientation to aid in the design of substrates forexperimental manipulation, such as in binding experiments, antibodysynthesis, etc. Secondary structural analysis can also be done toidentify regions of the Mam and/or a Mam-IP that assume specificstructures (Chou and Fasman, 1974, Biochemistry 13:222-223).Manipulation, translation, secondary structure prediction,hydrophilicity and hydrophobicity profiles, open reading frameprediction and plotting, and determination of sequence homologies, canbe accomplished using computer software programs available in the art.

Other methods of structural analysis including but not limited to X-raycrystallography (Engstrom, 1974, Biochem. Exp. Biol. 11:7-13), massspectroscopy and gas chromatography (see, Methods in Protein Science, J.Wiley and Sons, New York, 1997), and computer modeling (Fletterick andZoller, eds., 1986, Computer Graphics and Molecular Modeling, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor Press, New York) can also be employed.

5.2 Identification and Isolation of Mip30 and Mip6 Genes

The present invention relates to the nucleotide sequences encoding aMip30 or Mip6 protein. In specific embodiments, the Mip30, or Mip6nucleic acid sequence comprises the sequence of SEQ ID NOS:17 or 19,respectively, or a portion thereof, or a nucleotide sequence encoding,in whole or in part, a Mip30 or Mip6 protein (e.g., a protein comprisingthe amino acid sequence of SEQ ID NOS:18 or 20, respectively, or aportion thereof). The invention provides purified nucleic acidsconsisting of at least 8 nucleotides (i.e., a hybridizable portion) ofan Mip30, or Mip6 sequence. In other embodiments, the nucleic acidsconsist of at least about 25 (continuous) nucleotides, 50 nucleotides,100 nucleotides, 150 nucleotides, or 200 nucleotides of a Mip30 or Mip6gene sequence, or a full-length Mip30 or Mip6 gene sequence. In anotherembodiment, the nucleic acids are smaller than about 35, 200 or 500nucleotides in length. Nucleic acids can be single or double stranded.

The invention also relates to nucleic acids hybridizable to orcomplementary to the foregoing sequences, in particular the inventionprovides the inverse complement to nucleic acids hybridizable to theforegoing sequences (i.e., the inverse complement of a nucleic acidstrand has the complementary sequence running in reverse orientation tothe strand so that the inverse complement would hybridize withoutmismatches to the nucleic acid strand; thus, for example, where thecoding strand is hybridizable to a nucleic acid with no mismatchesbetween the coding strand and the hybridizable strand, then the inversecomplement of the hybridizable strand is identical to the codingstrand). In specific aspects, nucleic acid molecules are provided whichcomprise a sequence complementary to (specifically are the inversecomplement of) at least about 10, 25, 50, 100, or 200 nucleotides or theentire coding region of a Mip30 or Mip6 gene.

In a specific embodiment, a nucleic acid which is hybridizable to aMip30 or Mip6 nucleic acid sequence (e.g., having sequence SEQ ID NOS:17or 19, respectively), or to a nucleic acid sequence encoding a Mip30 orMip6 protein derivative (or a complement of the foregoing), underconditions of low stringency, is provided. By way of example and notlimitation, procedures using such conditions of low stringency are asfollows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA78:6789-6792): Filters containing DNA are pretreated for 6 hours at 40°C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denaturedsalmon sperm DNA. Hybridizations are carried out in the same solutionwith the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm³²P-labeled probe. Filters are incubated in hybridization mixture for18-20 hours at 40° C., and then washed for 1.5 hours at 55° C. in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Filters are blotted dry and exposed forautoradiography. If necessary, filters are washed for a third time at65-68° C. and reexposed to film. Other conditions of low stringencywhich may be used are well known in the art (e.g., as employed forcross-species hybridizations).

In another specific embodiment, a nucleic acid sequence which ishybridizable to an Mip30 or Mip6 nucleic acid sequence (or a complementof the foregoing) or to a nucleic acid sequence encoding a Mip30 or Mip6derivative under conditions of high stringency is provided. By way ofexample and not limitation, procedures using such conditions of highstringency are as follows: Prehybridization of filters containing DNA iscarried out for 8 hours to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 hours at 65° C. in prehybridization mixture containing100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeledprobe. Washing of filters is done at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This isfollowed by a wash in 0.1×SSC at 50° C. for 45 minutes beforeautoradiography. Other conditions of high stringency which may be usedare well known in the art.

In another specific embodiment, a nucleic acid sequence which ishybridizable to a Mip30 or Mip6 nucleic acid sequence or to a nucleicacid sequence encoding a Mip30 or Mip6 derivative (or a complement ofthe foregoing) under conditions of moderate stringency is provided. Forexample, but not limited to, procedures using such conditions ofmoderate stringency are as follows: Filters containing DNA arepretreated for 6 hours at 55° C. in a solution containing 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA.Hybridizations are carried out in the same solution with 5-20×10⁶ cpm³²P-labeled probe. Filters are incubated in hybridization mixture for18-20 hours at 55° C., and then washed twice for 30 minutes at 60° C. ina solution containing 1×SSC and 0.1% SDS. Filters are blotted dry andexposed for autoradiography. Other conditions of moderate stringencywhich may be used are well-known in the art. Washing of filters is doneat 37° C. for 1 hour in a solution containing 2×SSC, 0.1% SDS.

Nucleic acid molecules encoding derivatives and analogs of Mip30 or Mip6proteins (see this Section, supra), or Mip30 or Mip6 antisense nucleicacids (see Section 5.6.9, infra) are additionally provided. As isreadily apparent, as used herein, a “nucleic acid encoding a fragment orportion of a Mip30 or Mip6 protein” shall be construed as referring to anucleic acid encoding only the recited fragment or portion of the Mip30or Mip6 protein, and not the other contiguous portions of the Mip30 orMip6 as a continuous sequence.

Within nucleotide sequences, potential open reading frames can beidentified using the NCBI BLAST program ORF Finder available to thepublic. Because all known protein translation products are at least 60amino acids or longer (Creighton, 1992, Proteins, 2^(nd) Ed., W.H.Freeman and Co., New York), only those ORFs potentially encoding aprotein of 60 amino acids or more are considered. If an initiationmethionine codon (ATG) and a translational stop codon (TGA, TAA, or TGA)are identified, then the boundaries of the protein are defined. Otherpotential proteins include any open reading frames that extend to the5′end of the nucleotide sequence, in which case the open reading framepredicts the C-terminal or core portion of a longer protein. Similarly,any open reading frame that extends to the 3′ end of the nucleotidesequence predicts the N-terminal portion of a longer protein.

Any method available in the art can be used to obtain a full length(i.e., encompassing the entire coding region) cDNA clone encoding aMip30 or Mip6 protein. In particular, the polymerase chain reaction(PCR) can be used to amplify sequences in silico from a cDNA library.Oligonucleotide primers that hybridize to sequences at the 3′ and 5′termini of the identified sequences can be used as primers to amplify byPCR sequences from a nucleic acid sample (cDNA or DNA), preferably acDNA library, from an appropriate source (e.g., the sample from whichthe initial cDNA library for the modified yeast two hybrid assay fusionpopulation was derived).

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase. The DNA being amplified can include genomicDNA or cDNA sequences from any eukaryotic species. One can choose tosynthesize several different degenerate primers, for use in the PCRreactions. It is also possible to vary the stringency of hybridizationconditions used in priming the PCR reactions, to amplify nucleic acidhomologs (e.g., to obtain Mip30 or Mip6 sequences from species otherthan humans, or to obtain human sequences with homology to Mip30 orMip6) by allowing for greater or lesser degrees of nucleotide sequencesimilarity between the known nucleotide sequence and the nucleic acidhomolog being isolated. For cross species hybridization, low stringencyconditions are preferred. For same species hybridization, moderatelystringent conditions are preferred.

After successful amplification of the nucleic acid containing all or aportion of the Mip30 or Mip6 sequence, that segment may be molecularlycloned and sequenced, and utilized as a probe to isolate a complete cDNAor genomic clone. This, in turn, will permit the determination of thegene's complete nucleotide sequence, the analysis of its expression, andthe production of its protein product for functional analysis, asdescribed infra. In this fashion, the nucleotide sequence of the entireMip30 or Mip6 gene, as well as additional genes encoding a Mip30 or Mip6protein or analog may be identified.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the Mip30 or Mip6 gene. The nucleic acids canbe isolated from vertebrates, including mammalian, human, porcine,bovine, feline, avian, equine, canine, as well as additional primatesources, insects, plants, etc. The DNA may be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), bychemical synthesis, by cDNA cloning, or by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (see, for example,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II). Clones derived from genomic DNA may containregulatory and intronic DNA regions in addition to coding regions;clones derived from cDNA will contain only exon sequences. Whatever thesource, the gene should be molecularly cloned into a suitable vector forpropagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, for example, bysonication. The linear DNA fragments can then be separated according tosize by standard techniques, including but not limited to, agaroseand/or polyacrylamide gel electrophoresis, and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, a portion of the Mip30 or Mip6 gene (of any species)(e.g., a PCR amplification product obtained as described above, or anoligonucleotide having a sequence of a portion of the known nucleotidesequence) or its specific RNA, or a fragment thereof, may be purifiedand labeled, and the generated DNA fragments may be screened by nucleicacid hybridization to the labeled probe (Benton, W. and Davis, R., 1977,Science 196:180-182; Grunstein, M. And Hogness, D., 1975, Proc. Natl.Acad. Sci. U.S.A. 72:3961-3964). Those DNA fragments with substantialhomology to the probe will hybridize. It is also possible to identifythe appropriate fragment by restriction enzyme digestion(s) andcomparison of fragment sizes with those expected according to a knownrestriction map if such is available, or by DNA sequence analysis andcomparison to the known nucleotide sequence of Mip30 or Mip6. Furtherselection can be carried out on the basis of the properties of the gene.Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isolectric focusingbehavior, proteolytic digestion maps, or antigenic properties or abilityto bind Mam, as is known for Mip30 and Mip6. If an anti-Mip30 oranti-Mip6 antibody is available, the protein may be identified bybinding of labeled antibody to the putatively Mip30 or Mip6 synthesizingclones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.

An alternative to isolating the Mip30 or Mip6 cDNA includes, but is notlimited to, chemically synthesizing the gene sequence itself from aknown sequence. Other methods are possible and within the scope of theinvention.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives or the pBluescript vector(Stratagene, La Jolla, Calif.). Insertion into a cloning vector can, forexample, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically “polished” to ensure compatibility. Alternatively, anysite desired may be produced by ligating nucleotide sequences (linkers)onto the DNA termini; these ligated linkers may comprise specificchemically synthesized oligonucleotides encoding restrictionendonuclease recognition sequences. In an alternative method, thecleaved vector and the Mip30 or Mip6 gene may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated Mip30 or Mip6 gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

The Mip30 or Mip6 nuclear acid sequence provided by the presentinvention includes those nucleotide sequences encoding substantially thesame amino acid sequence as found in native Mip30 or Mip6 protein, andthose encoded amino acid sequences with functionally equivalent aminoacids, as well as those encoding other Mip30 or Mip6 derivatives oranalogs, as described in Section 5.1, supra, for Mip30 and Mip6derivatives and analogs.

5.3 Antibodies to Mam:Mam-IP Complexes, and Mip30 and Mip6 Proteins

According to the present invention, the Mam:Mam-IP complex (e.g., Mamcomplexed with Mip1, Mip30 or Mip6), or fragments, derivatives orhomologs thereof, or Mip30 or Mip6 protein or fragments, homologs andderivatives thereof, may be used as immunogens to generate antibodieswhich immunospecifically bind such immunogens. Such antibodies includebut are not limited to polyclonal, monoclonal, chimeric, and singlechain antibodies, Fab fragments, and Fab expression libraries. In aspecific embodiment, antibodies to complexes of human Mam and a humanMam-IP are produced. In another embodiment, complexes formed fromfragments of a Mam and a Mam-IP, where the fragments contain the proteindomain that interacts with the other member of the complex, are used asimmunogens for antibody production. In another specific embodiment,Mip30 or Mip6 proteins or fragments, derivatives, or homologs thereofare used as immunogens.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a Mam:Mam-IP complex, or to a derivative oranalog thereof, or to a Mip30 or Mip6 protein, or derivative, fragmentor analog thereof.

For production of the antibody, various host animals can be immunized byinjection with the native Mam:Mam-IP complex, or Mip30 or Mip6 protein,or a synthetic version, or a derivative of the foregoing, such as across-linked Mam:Mam-IP. Such host animals include but are not limitedto rabbits, mice, rats, etc. Various adjuvants can be used to increasethe immunological response, depending on the host species, and includebut are not limited to Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, and potentially useful human adjuvants such as bacilleCalmette-Guerin (CG) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed towards a Mam:Mam-IPcomplex or to a Mip30 or Mip6 protein, or derivatives, fragments oranalogs thereof, any technique that provides for the production ofantibody molecules by continuous cell lines in culture may be used. Twoconceptually unique approaches are currently available for theproduction of human monoclonal antibodies—the ‘hybridoma’ technique,based on the fusion of antibody-producing B lymphocytes withplasmacytoma cells or lymphoblastoid cell lines; and the use ofEpstein-Barr virus (EBV) to ‘immortalize’ antigen-specific human Blymphocytes. Such techniques include but are not restricted to thehybridoma technique originally developed by Kohler and Milstein (1975,Nature 256:495-497) (the cell lines are made by fusion of a mousemyeloma and mouse spleen cells from an immunised donor), the triomatechnique (Rosen et al., 1977, Cell 11:139-147), the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). In this technique, as in the hybridomaprocedure, it is important to use the blood lymphocytes of individualswho have previously been immunized with the antigens and have increasednumbers of specific antibody-producing cells. The procedure involves twosteps: (1) the enrichment of cells with receptors for the given antigen;and (2) ‘immortalization’ of these cells by EBV infection. In anadditional embodiment of the invention, monoclonal antibodies can beproduced in germ-free animals (See International Application No.PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cole et al., 1983,Proc. Natl. Acad. Sci. USA 80:2026-2030), or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, In: MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In fact,according to the invention, techniques developed for the production ofchimeric antibodies (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing the genes from a mouse antibodymolecule specific for the Mam:Mam-IP complex or Mip30 or Mip6 protein,together with genes from a human antibody molecule of appropriatebiological activity, can be used; such antibodies are within the scopeof this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce Mam:Mam-IP complex-specific and Mip30 or Mip6 protein-specificsingle chain antibody. An additional embodiment of the inventionutilizes techniques described for the construction of Fab expressionlibraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity for the Mam:Mam-IP complex, or an individual Mip30 or Mip6protein, derivative or analog. As reported by Huse et al., an Fabexpression library was constructed from mRNA isolated from a mouse thathad been immunized with the antigen NPN. The PCR amplification ofmessenger RNA isolated from spleen cells or hybridomas witholigonucleotides that incorporate restriction sites into the ends of theamplified product can be used to clone and express heavy and light chainsequences. Thus, the amplified fragments were cloned into a lambda phagevector in a predetermined reading frame for expression. Thecombinatorial library was constructed in two steps. In the first step,separate heavy and light chain libraries were constructed, and in thesecond step, these two libraries were used to construct a combinatoriallibrary by crossing them at the EcoRI site. After ligation, only clonesthat resulted from combination of a right arm of light chain-containingclones and a left arm of heavy chain-containing clones reconstituted aviable phage. After ligation and packaging, 2.5×10⁷ clones wereobtained. This is the combinatorial Fab expression library that wasscreened to identify clones having affinity for NPN. In an examinationof approximately 500 recombinant phage, approximately 60 percentcoexpressed light and heavy chain proteins. The light chain, heavy chainand Fab libraries were screened to determine whether they containedrecombinant phage that expressed antibody fragments binding NPN.Non-human antibodies can be humanized by known methods (e.g., see U.S.Pat. No. 5,225,539).

Antibody fragments that contain the idiotypes of a Mam:Mam-IP complex orof a Mip30 or Mip6 protein can be generated by techniques known in theart. For example, such fragments include but are not limited to: theF(ab)₂ fragment which can be produced by pepsin digestion of theantibody molecule; the Fab′ fragments that can be generated by reducingthe disulfide bridges of the F(ab)₂ fragment; the Fab fragments that canbe generated by treating the antibody molecular with papain and areducing agent; and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). To select antibodies specific to aparticular domain of the Mam:Mam-IP complex, or Mip30 or Mip6 protein,one may assay generated hybridomas for a product that binds to thefragment of the Mam:Mam-IP complex, or the Mip30 or Mip6 protein, thatcontains such a domain. For selection of an antibody that specificallybinds a Mam:Mam-IP complex but which does not specifically bind to theindividual proteins of the Mam:Mam-IP complex, one can select on thebasis of positive binding to the Mam:Mam-IP complex and a lack ofbinding to the individual Mam and Mam-IP proteins.

Antibodies specific to a domain of the Mam:Mam-IP complex are alsoprovided, as are antibodies to specific domains of the Mip30 or Mip6protein.

The foregoing antibodies can be used in methods known in the artrelating to the localization and/or quantitation of a Mam:Mam-IP complexor of a Mip30 or Mip6 protein of the invention, e.g., for imaging theseproteins, measuring levels thereof in appropriate physiological samples,in diagnostic methods, etc.

In another embodiment of the invention, anti-Mam:Mam-IP complexantibodies and fragments thereof, or anti-Mip30 or anti-Mpi6 antibodiesor fragments thereof, containing the binding domain, are therapeutics,see Section 5.6 below.

5.4 Methods for Identifying Modulators of Notch Signal Transduction orModulators of SUMO Conjugation Activity

The present invention is directed to methods of identifying a moleculethat alters Notch signal transduction in a cell comprising contactingthe cell with one or more candidate molecules; and measuring the amountof sumolation in the cell, wherein an increase or decrease in the amountof sumolation relative to said amount in a cell not so contacted withone or more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction. The present invention is alsodirected to methods of identifying a molecule that alters Notch signaltransduction in a cell comprising recombinantly expressing within thecell one or more candidate molecules; and measuring the amount ofsumolation in the cell, wherein an increase or decrease in the amount ofsumolation relative to said amount in a cell not so contacted with oneor more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction. The present invention is alsodirected to methods of identifying a molecule that alters Notch signaltransduction in a cell comprising microinjecting into the cell one ormore candidate molecules; and measuring the amount of sumolation in thecell, wherein an increase or decrease in the amount of sumolationrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules alterNotch signal transduction.

The present invention is directed to methods of identifying a moleculethat alters sumolation activity in a cell comprising contacting the cellwith one or more candidate molecules; and measuring the amount of Notchsignal transduction in the cell, wherein an increase or decrease in theamount of Notch signal transduction relative to said amount in a cellnot so contacted with one or more of the candidate molecules indicatesthat the candidate molecules alter sumolation activity. Another methodof identifying a molecule that alters sumolation in a cell comprisesrecombinantly expressing within the cell one or more candidatemolecules; and measuring the amount of Notch signal transduction in thecell, wherein an increase or decrease in the amount of Notch signaltransduction relative to said amount in a cell not so contacted with oneor more of the candidate molecules indicates that the candidatemolecules alter sumolation activity. Yet another method of identifying amolecule that alters sumolation activity in a cell comprisesmicroinjecting into the cell one or more candidate molecules; andmeasuring the amount of Notch signal transduction in the cell, whereinan increase or decrease in the amount of Notch signal transductionrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules altersumolation activity.

Methods that can be used to carrying out the foregoing are commonlyknown in the art and/or those methods disclosed herein. The cells usedin the methods of this embodiment can either endogenously orrecombinantly express Mam, Mip1, Mip30 and/or Mip6, or a fragment,derivative or analog thereof. Recombinant expression of a Mam and/orMam-IP is carried out by introducing the encoding nucleic acids intoexpression vectors and subsequently introducing the vectors into a cellto express the desired protein or simply introducing Mam and/or Mam-IPencoding nucleic acids into a cell for expression, as described inSection 5.2 or using procedures well known in the art. Nucleic acidsencoding Mam and Mip1 from a number of species have been cloned andsequenced and their expression is well known in the art. Illustrativeexamples of Mam and Mip molecules are set forth in FIGS. 1 and 6.Expression can be from expression vectors or intrachromosomal. In aspecific embodiment, standard human cell lines, such as HeLa cells andhuman kidney 293 cells, are employed in the screening assays.

Any method known to those of skill in the art for the insertion of Mamand/or Mam-IP-encoding DNA into a vector may be used to constructexpression vectors for expressing Mam and/or Mam-IP, including thosemethods described in Section 5.2, supra. In addition, a host cell strainmay be chosen which modulates the expression of Mam and/or Mam-IP, ormodifies and processes the gene product in the specific fashion desired.Expression from certain promoters can be elevated in the presence ofcertain inducers; thus, expression of the desired protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, cleavage) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the expressed desired protein.For example, expression in a bacterial system can be used to produce anunglycosylated core protein product. Expression in yeast will produce aglycosylated product. Expression in mammalian cells can be used toensure “native” glycosylation of a mammalian Mam and/or Mam-IP protein.

Sumolation, or SUMO conjugation activity, can be measured using methodswell know in the art, e.g., by an increase or decrease in theconjugation of SUMO to target proteins. The total cellular complement ofprotein targets or specific protein targets can be analyzed. The SUMOprotein can be introduced as a transgene in either an epitope-taggedform or an un-tagged form. Alternatively, the extent of endogenous SUMOconjugation activity can be assessed, e.g., using anti-SUMO antibodies,or by Western blot analysis in which the results would be amenable toquantification by densitometry. Further, since SUMO conjugation of aprotein often influences the intracellular localization of the protein,an assay based upon the localization of a specific target protein can beused. Also, since SUMO conjugation of a protein often stabilizes theprotein since SUMO competes with the same target lysine as ubiquitin,sumolation can be measured by measuring the stability, i.e., half-life,of the target protein, e.g., by Western blot analysis.

Notch signal transduction or Notch function can be measured using assayscommonly known in the art, e.g., by the ability of Notch to activatetranscription of a gene in the Enhancer of split complex, e.g., mγ, mδ,m5; or to activate transcription of vestigial, cut, or the HES1 gene. Anin vitro transcription assay utilizing HES1 has been described (Wu etal., 2000, Nature Genetics 26:484-489; Jarriault et al., 1995, Nature377:355-358). Thus, increased levels of mγ, mδ, m5, vestigial, cut orHES1 mRNA or protein indicates an increased level of Notch signaltransduction or Notch function. Conversely, decreased levels of mγ, mδ,m5, vestigial, cut or HES1 mRNA or protein indicates a decreased levelof Notch signal transduction or Notch function. Further, activation ofNotch signal transduction results in the inhibition of differentiationof precursor cells. See, U.S. Pat. No. 5,780,300. Thus, Notch signaltransduction can also be measured by assaying for differentiation ofprecursor cells. Maintenance of the differentiation state of theprecursor cell indicates active Notch signal transduction. A change inthe differentiation state of the precursor cell indicates inactive Notchsignal transduction. Additionally, reporter constructs with a reportergene under the control of a promoter containing a Notch-responsivepromoter element can also be used to detect Notch signal transduction.For example, the EBNA2 response element from the TP-1 promoter can beused in such a reporter construct.

5.4.1 Candidate Molecules

Any molecule known in the art can be tested for its ability to modulate(increase or decrease) Notch signal transduction or sumolation activityas detected by a change in the ability of a cell to differentiate or achange in HES1 expression (for Notch signal transduction) or by a changein levels of sumolation of cellular proteins or amount thereof (forsumolation activity). By way of example, a change in the level ofsumolation can be detected by detecting a change in the whether a testprotein is conjugated to SUMO. For identifying a molecule that modulatesNotch signal transduction or sumolation, candidate molecules can bedirectly provided to a cell or, in the case of candidate proteins, canbe provided by providing their encoding nucleic acids under conditionsin which the nucleic acids are recombinantly expressed to produce thecandidate proteins within the cell.

This embodiment of the invention is well suited to screen chemicallibraries for molecules which modulate, e.g., inhibit, antagonize, oragonize Notch signal transduction or sumolation activity. The chemicallibraries can be peptide libraries, peptidomimetic libraries, chemicallysynthesized libraries, recombinant, e.g., phage display libraries, andin vitro translation-based libraries, other non-peptide syntheticorganic libraries, etc.

Exemplary libraries are commercially available from several sources(ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In some cases,these chemical libraries are generated using combinatorial strategiesthat encode the identity of each member of the library on a substrate towhich the member compound is attached, thus allowing direct andimmediate identification of a molecule that is an effective modulator.Thus, in many combinatorial approaches, the position on a plate of acompound specifies that compound's composition. Also, in one example, asingle plate position may have from 1-20 chemicals that can be screenedby administration to a well containing the interactions of interest.Thus, if modulation is detected, smaller and smaller pools ofinteracting pairs can be assayed for the modulation activity. By suchmethods, many candidate molecules can be screened.

Many diversity libraries suitable for use are known in the art and canbe used to provide compounds to be tested according to the presentinvention. Alternatively, libraries can be constructed using standardmethods. Chemical (synthetic) libraries, recombinant expressionlibraries, or polysome-based libraries are exemplary types of librariesthat can be used.

The libraries can be constrained or semirigid (having some degree ofstructural rigidity), or linear or nonconstrained. The library can be acDNA or genomic expression library, random peptide expression library ora chemically synthesized random peptide library, or non-peptide library.Expression libraries are introduced into the cells in which the assayoccurs, where the nucleic acids of the library are expressed to producetheir encoded proteins.

In one embodiment, peptide libraries that can be used in the presentinvention may be libraries that are chemically synthesized in vitro.Examples of such libraries are given in Houghten et al., 1991, Nature354:84-86, which describes mixtures of free hexapeptides in which thefirst and second residues in each peptide were individually andspecifically defined; Lam et al., 1991, Nature 354:82-84, whichdescribes a “one bead, one peptide” approach in which a solid phasesplit synthesis scheme produced a library of peptides in which each beadin the collection had immobilized thereon a single, random sequence ofamino acid residues; Medynski, 1994, Bio/Technology 12:709-710, whichdescribes split synthesis and T-bag synthesis methods; and Gallop etal., 1994, J. Medicinal Chemistry 37(9):1233-1251. Simply by way ofother examples, a combinatorial library may be prepared for use,according to the methods of Ohlmeyer et al., 1993, Proc. Natl. Acad.Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; orSalmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712. PCTPublication No. WO 93/20242 and Brenner and Lerner, 1992, Proc. Natl.Acad. Sci. USA 89:5381-5383 describe “encoded combinatorial chemicallibraries,” that contain oligonucleotide identifiers for each chemicalpolymer library member.

In a preferred embodiment, the library screened is a biologicalexpression library that is a random peptide phage display library, wherethe random peptides are constrained (e.g., by virtue of having disulfidebonding).

Further, more general, structurally constrained, organic diversity(e.g., nonpeptide) libraries, can also be used. By way of example, abenzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad.Sci. USA 91:4708-4712) may be used.

Conformationally constrained libraries that can be used include but arenot limited to those containing invariant cysteine residues which, in anoxidizing environment, cross-link by disulfide bonds to form cystines,modified peptides (e.g., incorporating fluorine, metals, isotopiclabels, are phosphorylated, etc.), peptides containing one or morenon-naturally occurring amino acids, non-peptide structures, andpeptides containing a significant fraction of γ-carboxyglutamic acid.

Libraries of non-peptides, e.g., peptide derivatives (for example, thatcontain one or more non-naturally occurring amino acids) can also beused. One example of these are peptoid libraries (Simon et al., 1992,Proc. Natl. Acad. Sci. USA 89:9367-9371). Peptoids are polymers ofnon-natural amino acids that have naturally occurring side chainsattached not to the alpha carbon but to the backbone amino nitrogen.Since peptoids are not easily degraded by human digestive enzymes, theyare advantageously more easily adaptable to drug use. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al., 1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The members of the peptide libraries that can be screened according tothe invention are not limited to containing the 20 naturally occurringamino acids. In particular, chemically synthesized libraries andpolysome based libraries allow the use of amino acids in addition to the20 naturally occurring amino acids (by their inclusion in the precursorpool of amino acids used in library production). In specificembodiments, the library members contain one or more non-natural ornon-classical amino acids or cyclic peptides. Non-classical amino acidsinclude but are not limited to the D-isomers of the common amino acids,α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid;γ-Abu, ε-Ahx, 6-amino hexanoic acid; Aib, 2-amino isobutyric acid;3-amino propionic acid; ornithine; norleucine; norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, designeramino acids such as β-methyl amino acids, Cα-methyl amino acids,Nα-methyl amino acids, fluoro-amino acids and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

In a specific embodiment, fragments and/or analogs of Mam or Mip1,especially peptidomimetics, are screened for activity as competitive ornon-competitive inhibitors of Notch signal transduction or sumolationactivity.

In another embodiment of the present invention, combinatorial chemistrycan be used to identify modulators of Notch signal transduction orsumolation activity. Combinatorial chemistry is capable of creatinglibraries containing hundreds of thousands of compounds, many of whichmay be structurally similar. While high throughput screening programsare capable of screening these vast libraries for affinity for knowntargets, new approaches have been developed that achieve libraries ofsmaller dimension but which provide maximum chemical diversity. (Seee.g., Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).

One method of combinatorial chemistry, affinity fingerprinting, haspreviously been used to test a discrete library of small molecules forbinding affinities for a defined panel of proteins. The fingerprintsobtained by the screen are used to predict the affinity of theindividual library members for other proteins or receptors of interest(in the instant invention, e.g., Mip1). The fingerprints are comparedwith fingerprints obtained from other compounds known to react with theprotein of interest to predict whether the library compound mightsimilarly react. For example, rather than testing every ligand in alarge library for interaction with Mip1, only those ligands having afingerprint similar to other compounds known to have that activity couldbe tested. (See, e.g., Kauvar et al., 1995, Chemistry and Biology2:107-118; Kauvar, 1995, Affinity fingerprinting, PharmaceuticalManufacturing International. 8:25-28; and Kauvar, Toxic-ChemicalDetection by Pattern Recognition in New Frontiers in AgrochemicalImmunoassay, D. Kurtz. L. Stanker and J. H. Skerritt. Editors, 1995,AOAC: Washington, D.C., 305-312).

Kay et al., 1993, Gene 128:59-65 (Kay) discloses a method ofconstructing peptide libraries that encode peptides of totally randomsequence that are longer than those of any prior conventional libraries.The libraries disclosed in Kay encode totally synthetic random peptidesof greater than about 20 amino acids in length. Such libraries can beadvantageously screened to identify modulators of Notch signaltransduction or sumolation activity. (See also U.S. Pat. No. 5,498,538dated Mar. 12, 1996; and PCT Publication No. WO 94/18318 dated Aug. 18,1994).

A comprehensive review of various types of peptide libraries can befound in Gallop et al., 1994, J. Med. Chem. 37:1233-1251.

5.5 Diagnostic, Prognostic, and Screening Uses of Mam:Mam-IP Complexesand Nucleic Acids, and Mip30 and Mip6 Proteins and Nucleic Acids

Mam:Mam-IP complexes (particularly Mam complexed with one of thefollowing: Mip1, Mip30 or Mip6) may be markers of normal physiologicalprocesses including, but not limited to, the physiological processesincluding signal transduction, cell fate and differentiation and mitoticevents, such as chromosomal segregation, and thus have diagnosticutility. Further, definition of particular groups of patients withelevations or deficiencies of a Mam:Mam-IP complex, or a Mip30 or Mip6protein, can lead to new classifications of diseases, furtheringdiagnostic ability.

Detecting levels of Mam:Mam-IP complexes, or individual proteins thathave been shown to form complexes with Mam, or the Mip30 or Mip6proteins; or detecting levels of mRNAs encoding components of theMam:Mam-IP complexes, or mRNAs encoding the Mip30 or Mip6 protein, maybe used in prognosis, to follow the course of disease state, to followtherapeutic response, etc.

Mam:Mam-IP complexes and the individual components of the Mam:Mam-IPcomplexes (e.g., Mam, Mip1, Mip30, Mip6), and derivatives, analogs andsubsequences thereof; Mam and/or Mam-IP, or Mip30 or Mip6 nucleic acids(and sequences complementary thereto); anti-Mam:Mam-IP complexantibodies and antibodies directed against the individual componentsthat can form Mam:Mam-IP complexes; and anti-Mip30 or anti-Mip6antibodies, have uses in diagnostics. Such molecules can be used inassays, such as immunoassays, to detect, prognose, diagnose, or monitorvarious conditions, diseases, and disorders, and treatment thereof,characterized by aberrant levels of Mam:Mam-IP complexes, or by aberrantlevels of Mip30 or Mip6 protein.

In particular, such an immunoassay is carried out by a method comprisingcontacting a sample derived from a patient with an anti-Mam:Mam-IPcomplex antibody, or an anti-Miup30 or anti-Mip6 antibody underconditions such that immunospecific binding can occur, and detecting ormeasuring the amount of any immunospecific binding by the antibody. In aspecific aspect, such binding of antibody, in tissue sections, can beused to detect aberrant Mam:Mam-IP complex formation, or aberrant Mip30or Mip6 protein localization, or aberrant (e.g., high, low or absent)levels of Mam:Mam-IP complex or complexes, or aberrant levels of Mip30or Mip6 protein. In a specific embodiment, an antibody against aMam:Mam-IP complex can be used to assay a patient tissue or serum samplefor the presence of the Mam:Mam-IP complex, where an aberrant level ofthe Mam:Mam-IP complex is an indication of a disease condition. Inanother embodiment, an antibody against Mip30 or Mip6 can be used toassay a patient tissue or serum sample for the presence of Mip30 or Mip6where an aberrant level of Mip30 or Mip6 is an indication of a diseasecondition. By “aberrant levels” is meant increased or decreased levelsrelative to that present, or a standard level representing that present,in an analogous sample from a portion of the body or from a subject nothaving the disorder.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such asWestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, and protein A immunoassays, to namebut a few.

Nucleic acids encoding the components of the Mam:Mam-IP complexes (e.g.,Mam, Mip1, Mip30 or Mip6) and nucleic acids encoding a Mip30 or Mip6protein, and related nucleotide sequences and subsequences, includingcomplementary sequences, can also be used in hybridization assays. TheMam and/or Mam-IP nucleotide sequence, or a subsequence thereof,comprising about at least 8 nucleotides, can be used as hybridizationprobes. Hybridization assays can be used to detect, prognose, diagnose,or monitor conditions, disorders, or disease states associated withaberrant levels of the mRNAs encoding the components of a Mam:Mam-IPcomplex, or a Mip30 or Mip6 protein, as described supra. In particular,such a hybridization assay is carried out by a method comprisingcontacting a sample containing nucleic acid with a nucleic acid probecapable of hybridizing to Mam or a Mam-IP DNA or RNA, under conditionssuch that hybridization can occur, and detecting or measuring anyresulting hybridization. In a preferred aspect, the hybridization assayis carried out using nucleic acid probes capable of hybridizing to Mamand to a binding partner of Mam to measure concurrently the expressionof both members of a Mam:Mam-IP complex. In another preferredembodiment, the expression of mRNAs encoding Mip30 or Mip6 is measured.

In specific embodiments, diseases and disorders involving orcharacterized by aberrant levels of Mam:Mam-IP complexes (e.g.,complexes of Mam with Mip1, Mip30 or Mip6 protein) can be diagnosed, ortheir suspected presence can be screened for, or a predisposition todevelop such disorders can be detected, by detecting aberrant levels ofa Mam:Mam-IP complex, or un-complexed Mam and/or a Mam-IP protein ornucleic acids or functional activity, including but not restricted to,binding to an interacting partner, or by detecting mutations in Mamand/or in a Mam-IP RNA, DNA or protein (e.g., translocations,truncations, changes in nucleotide or amino acid sequence relative towild-type Mam and/or Mam-IP) that cause increased or decreasedexpression or activity of a Mam:Mam-IP complex and/or Mam and/or proteinthat binds to Mam. Such diseases and disorders include but are notlimited to those described in Section 5.6 and its subsections.

By way of example, levels of a Mam:Mam-IP complex or the individualcomponents of a Mam:Mam-IP complex can be detected by immunoassay;levels of Mam and/or of Mam-IP mRNA can be detected by hybridizationassays (e.g., Northern blots, dot blots); binding of Mam or to a Mam-IPcan be measured by binding assays commonly known in the art,translocations and point mutations in Mam and/or in genes encoding aMam-IP can be detected by Southern blotting, RFLP analysis, PCR usingprimers that preferably generate a fragment spanning at least most ofthe Mam and/or Mam-IP gene, sequencing of the Mam and/or Mam-IP genomicDNA or cDNA obtained from the patient, etc.

Assays well known in the art (e.g., assays described above such asimmunoassays, nucleic acid hybridization assays, activity assays, etc.)can be used to determine whether one or more particular Mam:Mam-IPcomplexes are present at either increased or decreased levels, or areabsent, in samples from patients suffering from a particular disease ordisorder, or having a predisposition to develop such a disease ordisorder as compared to the levels in samples from subjects not havingsuch a disease or disorder.

Additionally, these assays can be used to determine whether the ratio ofthe Mam:Mam-IP complex to the un-complexed components of the Mam:Mam-IPcomplex, i.e., Mam and/or the specific Mam-IP in the complex ofinterest, is increased or decreased in samples from patients sufferingfrom a particular disease or disorder, or having a predisposition todevelop such a disease or disorder, as compared to the ratio in samplesfrom subjects not having such a disease or disorder.

In the event that levels of one or more particular Mam:Mam-IP complexesare determined to be increased in patients suffering from a particulardisease or disorder, or having a predisposition to develop such adisease or disorder, then the particular disease or disorder orpredisposition for a disease or disorder can be diagnosed, haveprognosis defined for, be screened for, or be monitored by detectingincreased levels of the one or more Mam:Mam-IP complexes, the mRNA thatencodes the members of the one or more particular Mam:Mam-IP complexes,or Mam:Mam-IP complex functional activity.

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving increased levels of one or more Mam:Mam-IP complexescan be diagnosed, or their suspected presence can be screened for, or apredisposition to develop such disorders can be detected, by detectingincreased levels of the one or more Mam:Mam-IP complexes, the mRNAencoding both members of the complex, or complex functional activity, orby detecting mutations in Mam or the Mam-IP (e.g., translocations innucleic acids, truncations in the gene or protein, changes in nucleotideor amino acid sequence relative to wild-type Mam or Mam-IP) thatstabilize or increase Mam:Mam-IP complex formation.

In the event that levels of one or more particular Mam:Mam-IP complexesare determined to be decreased in patients suffering from a particulardisease or disorder or having a predisposition to develop such a diseaseor disorder, then the particular disease or disorder or predispositionfor a disease or disorder can be diagnosed, have its prognosisdetermined, be screened for, or be monitored by detecting decreasedlevels of the one or more Mam:Mam-IP complexes, the mRNA that encodesthe members of the particular one or more Mam:Mam-IP complexes, orMam:Mam-IP complex functional activity.

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving decreased levels of one or more Mam:Mam-IP complexescan be diagnosed, or their suspected presence can be screened for, or apredisposition to develop such disorders can be detected, by detectingdecreased levels of the one or more Mam:Mam-IP complexes, the mRNAencoding the members of the one or more complexes, or complex functionalactivity, or by detecting mutations in Mam or the Mam-IP (e.g.,translocations in nucleic acids, truncations in the gene or protein,changes in nucleotide or amino acid sequence relative to wild-type Mamor the Mam-IP) that inhibit or reduce Mam:Mam-IP complex formation.

In another specific embodiment, diseases and disorders involvingaberrant expression of a Mip30 or Mip6 protein are diagnosed, or theirsuspected presence can be screened for, or a predisposition to developsuch disorders can be detected, by detecting aberrant levels of a Mip30or Mip6 protein, or mRNA, or functional activity, or by detectingmutations in a Mip30 or Mip6 protein or mRNA or DNA (e.g.,translocations in nucleic acids, truncations in the gene or protein,changes in nucleotide or amino acid sequence relative to wild-type Mip30or Mip6) that cause aberrant expression or activity of Mip30 or Mip6protein. Such diseases and disorders include but are not limited tothose described infra, Section 5.6. By way of example, levels of Mip30or Mip6 mRNA or protein, Mam binding activity, or the presence oftranslocations or point mutations, can be determined as described above.

Assays well known in the art (e.g., assays described above such asimmunoassays, nucleic acid hybridization assays, activity assays, etc.)can be used to determine whether Mip30 or Mip6 are present at eitherincreased or decreased levels, or are absent, in samples from patientssuffering from a particular disease or disorder, or having apredisposition to develop such a disease or disorder, as compared to thelevels in samples from subjects not having such a disease or disorder.

In the event that levels of Mip30 or Mip6 are determined to be increasedin patients suffering from a particular disease or disorder, or having apredisposition to develop such a disease or disorder, then theparticular disease or disorder or predisposition for a disease ordisorder can be diagnosed, have its prognosis determined, be screenedfor, or be monitored by detecting increased levels of Mip30 or Mip6protein or mRNA, or Mip30 or Mip6 functional activity (e.g., binding toMam).

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving increased levels of a Mip30 or Mip6 protein can bediagnosed, or their suspected presence can be screened for, or apredisposition to develop such disorders can be detected, by detectingincreased levels of a Mip30 or Mip6 protein or encoding nucleic acids,or Mip30 or Mip6 functional activity, or by detecting mutations in Mip30or Mip6 (e.g., translocations in nucleic acids, truncations in the geneor protein, changes in nucleotide or amino acid sequence relative towild-type Mip30 or Mip6) that enhance Mip30 or Mip6 stability orfunctional activity.

In the event that levels of Mip30 or Mip6 are determined to be decreasedin patients suffering from a particular disease or disorder or having apredisposition to develop such a disease or disorder, then theparticular disease or disorder or predisposition for a disease ordisorder can be diagnosed, or prognosis determined, be screened for, orbe monitored by detecting decreased levels of the Mip30 or Mip6 proteinsor nucleic acids, or Mip30 or Mip6 functional activity.

Accordingly, in a specific embodiment of the invention, diseases anddisorders involving decreased levels of Mip30 or Mip6 can be diagnosed,or their suspected presence can be screened for, or a predisposition todevelop such disorders can be detected, by detecting decreased levels ofMip30 or Mip6 protein or nucleic acids, or Mip30 or Mip6 functionalactivity, or by detecting mutations in Mip30 or Mip6 (e.g.,translocations in nucleic acids, truncations in the gene or protein,changes in nucleotide or amino acid sequence relative to wild-type Mip30or Mip6) that destabilize or reduce Mip30 or Mip6 functional activity.

The use of detection techniques, especially those involving antibodiesagainst Mam:Mam-IP complexes, or against a Mip30 or Mip6 protein,provides a method of detecting specific cells that express the complexor protein. Using such assays, specific cell types can be defined inwhich one or more particular Mam:Mam-IP complex, or Mip30 or Mip6protein, is expressed, and the presence of the complex or protein can becorrelated with cell viability.

Also embodied are methods to detect a Mam:Mam-IP complex, or a Mip30 orMip6 protein, in cell culture models that express particular Mam:Mam-IPcomplexes or Mip30 or Mip6 proteins, or derivatives thereof, for thepurpose of characterizing or preparing Mam:Mam-IP complexes, or Mip30 orMip6 proteins for harvest. This embodiment includes cell sorting ofprokaryotes such as, but not restricted to, bacteria (Davey and Kell,1996, Microbiol. Rev. 60: 641-696), primary cultures and tissuespecimens from eukaryotes, including mammalian species such as human(Steele et al., 1996, Clin. Obstet. Gynecol 39:801-813), and continuouscell cultures (Orfao and Ruiz-Arguelles, 1996, Clin. Biochem. 29:5-9).Such isolations can also be used as methods of diagnosis, describedsupra.

Kits for diagnostic use are also provided that comprise in one or morecontainers an anti-Mam:Mam-IP complex antibody or an anti-Mip30 oranti-Mip6 antibody, and, optionally, a labeled binding partner to theantibody. Alternatively, the anti-Mam:Mam-IP complex antibody, oranti-Mip30 or anti-Mip6 antibody, can be labeled with a detectablemarker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactivemoiety. A kit is also provided that comprises in one or more containersa nucleic acid probe capable of hybridizing to Mam and/or a Mam-IP(e.g., Mip1, Mip30, Mip6) mRNA. In a specific embodiment, a kit cancomprise in one or more containers a pair of primers (e.g., each in thesize range of about 6-30 nucleotides) that are capable of primingamplification [e.g., by polymerase chain reaction (see e.g., Innis etal., 1990, PCR Protocols, Academic Press, Inc., San Diego, Calif.),ligase chain reaction (see EP 320,308), use of Qβ replicase, cyclicprobe reaction, or other methods known in the art], under appropriatereaction conditions of at least a portion of a Mam and/or a Mam-IP, oran Mip30 or Mip6 nucleic acid sequence. A kit can optionally furthercomprise in a container a predetermined amount of a purified Mam:Mam-IPcomplex, Mam and/or a Mam-IP, or a Mip30 or Mip6 protein or an encodingnucleic acid molecule thereof, e.g. for use as a standard or control.

5.6 Therapeutic Uses of Mam:Mam-IP Complexes and Mip30 and Mip6

5.6.1 Therapeutic Uses of Mam and Mam-Interactants

The invention provides for treatment or prevention of various diseasesand disorders by administration of a therapeutic compound (termed herein“Therapeutic”). Such “Therapeutics” include but are not limited to:Mam:Mam-IP complexes (e.g., Mam complexed with Mip1, Mip30 or Mip6), Mamand the individual Mam-IP proteins and analogs and derivatives(including fragments) of the foregoing (e.g., as described hereinabove); antibodies there to (as described herein above); nucleic acidsencoding Mam and/or a Mam-IP, and analogs or derivatives thereof (e.g.,as described herein above); Mam and/or Mam-IP antisense nucleic acids,and Mam:Mam-IP complex and Mip30 and Mip6 modulators (i.e., inhibitors,agonists and antagonists).

As reviewed in Section 2, supra, Mam is centrally implicated inphysiological processes, including but not limited to, signaltransduction, and cell fate and differentiation. Likewise, Mam has beenstrongly implicated in pathological conditions, including but notlimited to, and cancer. The Mam interactant Mip1, described in thepresent invention, is involved in mitosis, telomere regulation, andchromosome segregation, see section 2, supra.

Disorders of cell cycle progression, cell differentiation, andtranscriptional control, including cancer and tumorigenesis and tumorprogression can involve Mam and particularly the interactants Mip1,Mip30 and Mip6. The effect of the Mip1 protein on tumorigenesis may bedue to the involvement of aberrant mitotic events in cancer.

Interactants Mip30 and Mip6 show no overall homologies to knownproteins. However, Mip30 contains seven C2H2 zinc-finger repeats, whichmay be involved in protein-protein interactions, a HMG-1 and HMG-YDNA-binding domain (A+T-hook), and a bipartite nuclear localizationsignal. The only identifiable motif in the Mip6 protein is a bipartitenuclear localization signal (amino acids 420-437).

5.6.2 Treatment of Diseases and Disorders with Increased Mam:Mam-IPComplexes

A wide range of cell diseases affected by intracellular signaltransduction, and chromosome segregation can be treated or prevented byadministration of a Therapeutic that modulates (i.e., inhibits,antagonizes, enhances or promotes) Mam:Mam-IP complex activity. All ofthese disorders can be treated or prevented by administration of aTherapeutic that modulates (i.e., inhibits, antagonizes, enhances orpromotes) Mam:Mam-IP complex activity, or modulates Mip30 or Mip6activity.

Diseases or disorders associated with aberrant levels of Mam:Mam-IPcomplex levels or activity, or aberrant levels of Mip30 or Mip6, may betreated by administration of a Therapeutic that modulates Mam:Mam-IPcomplex formation or activity, or Mip30 or Mip6 activity. In a specificembodiment, the activity or level of Mam is modulated by administrationof a Mam-IP. In another specific embodiment, the activity or level of aMam-IP is modulated by administration of Mam.

5.6.2.1 Antagonizing the Complex Formation or Activity

Diseases and disorders characterized by increased (relative to a subjectnot suffering from the disease or disorder) Mam:Mam-IP levels oractivity, or increased Mip30 or Mip6 levels or activity, can be treatedwith Therapeutics that antagonize (i.e., reduce or inhibit) Mam:Mam-IPcomplex formation or activity, or Mip30 or Mip6 levels or activity.Therapeutics that can be used, include but are not limited to, Mam or aMam-IP, or analogs, derivatives or fragments thereof; anti-Mam:Mam-IPcomplex antibodies (e.g., antibodies specific for Mam:Mip1, Mam:Mip30,Mam:Mip6 complexes), and anti-Mip30 or anti-Mip6 antibodies, fragmentsand derivatives thereof containing the binding region thereof; nucleicacids encoding Mam or a Mam-IP; concurrent administration of Mam andMam-IP antisense nucleic acids, or Mip30 or Mip6 antisense nucleicacids, or Mam and/or Mam-IP, or Mip30 or Mip6 nucleic acids that aredysfunctional (e.g., due to a heterologous (non-Mam and/or non-Mam-IP,or non-Mip30 or non-Mip6) insertion within the coding sequences of theMam coding sequences)) that are used to “knockout” endogenous Mam and/orMam-IP function by homologous recombination (see, e.g., Capecchi, 1989,Science 244:1288-1292).

In a specific embodiment of the invention, a nucleic acid containing aportion of a Mam and/or a Mam-IP gene in which the Mam and/or Mam-IPsequences flank (are both 5′ and 3′ to) a different gene sequence, isused as a Mam and/or a Mam-IP antagonist, or to promote Mam and/orMam-IP inactivation by homologous recombination (see also Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935, Zijlstra etal., 1989, Nature 342:435-438). Additionally, mutants or derivatives ofa first Mam-IP protein that have greater affinity for Mam than a secondMam-IP may be administered to compete with the second Mam-IP protein forMam binding, thereby reducing the levels of Mam complexes with thesecond Mam-IP. Other Therapeutics that inhibit Mam:Mam-IP complex orMip30 or Mip6 function can be identified by use of known convenient invitro assays, e.g., based on their ability to inhibit Mam:Mam-IP bindingor as described in Section 5.8 infra.

In specific embodiments, Therapeutics that antagonize Mam:Mam-IP complexformation or activity, or a Mip30 or Mip6 activity, are administeredtherapeutically (including prophylactically): (1) in diseases ordisorders involving an increased (relative to normal or desired) levelof Mam:Mam-IP complex, or a Mip30 or Mip6 protein, for example, inpatients where a Mam:Mam-IP complex or a Mip30 or Mip6 protein isoveractive or overexpressed; or (2) in diseases or disorders wherein invitro (or in vivo) assays (see infra) indicate the utility of aMam:Mam-IP complex or Mip30 or Mip6 antagonist administration. Increasedlevels of Mam:Mam-IP complexes or increased Mip30 or Mip6 proteinlevels, can be readily detected, e.g., by quantifying protein and/orRNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) andassaying it in vitro for RNA or protein levels, structure and/oractivity of the expressed Mam:Mam-IP complex (or the Mam and Mam-IPmRNA), or the Mip30 or Mip6 protein or mRNA levels. Many methodsstandard in the art can be thus employed, including but not limited to:immunoassays to detect and/or visualize Mam:Mam-IP complexes, or Mip30or Mip6 protein (e.g., Western blot, immunoprecipitation followed bysodium dodecyl sulfate polyacrylamide gel electrophoresis,immunocytochemistry, etc.) and/or hybridization assays to detectconcurrent expression of Mam and a Mam-IP, or individual Mip30 or Mip6mRNA (e.g., Northern blot assays, dot blots, in situ hybridization,etc.).

5.6.2.2 Reducing the Complex Expression

A more specific embodiment includes methods of reducing Mam:Mam-IPcomplex expression (i.e., the expression of the two components of theMam:Mam-IP complex and/or formation of the complex), or reducing Mip30or Mip6 expression, by targeting mRNAs that express the proteinmoieties. RNA therapeutics currently fall within three classes,antisense species, ribozymes, or RNA aptamers (Good et al., 1997, GeneTherapy 4:45-54).

Antisense oligonucleotides have been the most widely used. By way ofexample, but not for limitation, antisense oligonucleotide methodologyto reduce Mam:Mam-IP complex formation is presented below in Subsection5.6.8. Ribozyme therapy involves the administration, induced expression,etc., of small RNA molecules with enzymatic ability to cleave, bind, orotherwise inactivate specific RNAs to reduce or eliminate expression ofparticular proteins (Grassi and Marini, 1996, Annals of Medicine28:499-510, Gibson, 1996, Cancer and Metastasis Reviews 15:287-299). Atpresent, the design of hairpin and hammerhead RNA ribozymes is necessaryto specifically target a particular mRNA, such as the mRNA encoding Mam.RNA aptamers are specific RNA ligands for proteins, such as for Tat andRev RNA (Good et al., 1997, Gene Therapy 4:45-54) that can specificallyinhibit their translation. Aptamers specific for Mam or a Mam-IP can beidentified by many methods well known in the art, for example but notlimited to the protein-protein interaction assay described in Section5.8.1 infra.

In another embodiment, the activity or level of Mam is reduced byadministration of a Mam-IP, or a nucleic acid that encodes a Mam-IP, orantibody that immunospecifically binds to a Mam-IP, or a fragment or aderivative of the antibody containing the binding domain thereof.Additionally, the level or activity of a Mam-IP may be reduced byadministration of a Mam or a Mam-IP nucleic acid, or an antibody thatimmunospecifically binds Mam, or a fragment or derivative of theantibody containing the binding domain thereof.

In another aspect of the invention, diseases or disorders associatedwith increased levels of Mam or a particular Mam-IP (e.g., Mip1, Mip30,Mip6) may be treated or prevented by administration of a Therapeuticthat increases Mam:Mam-IP complex formation, if the complex formationacts to reduce or inactivate Mam or the particular Mam-IP throughMam:Mam-IP complex formation. Such diseases or disorders can be treatedor prevented by administration of one member of the Mam:Mam-IP complex,including mutants of a member of the Mam:Mam-IP that have increasedaffinity for the other member of the Mam:Mam-IP complex (to causeincreased complex formation), administration of antibodies or othermolecules that stabilize the Mam:Mam-IP complex, etc.

5.6.3 Treatment of Diseases and Disorders Associated with UnderexpressedMam:Mam-IP Complexes

Diseases and disorders associated with underexpression of a Mam:Mam-IPcomplex, or Mam or a particular Mam-IP, are treated or prevented byadministration of a Therapeutic that promotes (i.e., increases orsupplies) Mam:Mam-IP complexes or function. Examples of such aTherapeutic include but are not limited to Mam:Mam-IP complexes andderivatives, analogs and fragments thereof that are functionally active(e.g., active to form Mam:Mam-IP complexes), un-complexed Mam and Mam-IPproteins, and derivatives, analogs, and fragments thereof, and nucleicacids encoding the members of a Mam:Mam-IP complex, or functionallyactive derivatives or fragments thereof (e.g., for use in gene therapy).In a specific embodiment are derivatives, homologs or fragments of Mamand/or a Mam-IP that increase and/or stabilize Mam:Mam-IP complexformation. Examples of other agonists can be identified using in vitroassays or animal models, examples of which are described supra, and inSection 5.10, infra.

5.6.3.1 Promotion of the Complex Function

In specific embodiments, Therapeutics that promote Mam:Mam-IP complexfunction, or promote Mip30 or Mip6 function, are administeredtherapeutically (including prophylactically): (1) in diseases ordisorders involving an absence or decreased (relative to normal ordesired) level of Mam:Mam-IP complex, or a Mip30 or Mip6 protein, forexample, in patients where Mam:Mam-IP complexes (or the individualcomponents necessary to form the complexes), or where Mip30 or Mip6protein is lacking, genetically defective, biologically inactive orunderactive, or under-expressed; or (2) in diseases or disorders whereinin vitro (or in vivo) assays (see infra) indicate the utility ofMam:Mam-IP complex, or Mip30 or Mip6 agonist administration. The absenceor decreased level of Mam:Mam-IP complex, or Mip30 or Mip6 protein orfunction, can be readily detected, e.g., by obtaining a patient tissuesample (e.g., from biopsy tissue) and assaying in vitro for RNA proteinlevels, activity of the expressed Mam:Mam-IP complex (or for theconcurrent expression of mRNA encoding the two components of theMam:Mam-IP complex), or Mip30 or Mip6 RNA, protein or activity. Manymethods standard in the art can be thus employed, including but notlimited to immunoassays to detect and/or visualize Mam:Mam-IP complexes(or the individual components of Mam:Mam-IP complexes), or Mip30 or Mip6protein (e.g., Western blot, immunoprecipitation followed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry,etc.) and/or hybridization assays to detect expression of the mRNAencoding the individual protein components of the Mam:Mam-IP complexesby detecting and/or visualizing Mam and a Mam-IP mRNA concurrently orseparately using, e.g., Northern blot assays, dot blots, in situhybridization, etc.

5.6.3.2 Increasing Mam or Mam-IP Levels

In a specific embodiment, the activity or level of Mam is increased byadministration of a Mam-IP, or derivative or analog thereof, a nucleicacid encoding a Mam-IP, or an antibody that immunospecifically binds aMam-IP, or a fragment or derivative of the antibody contains the bindingdomain thereof. In another specific embodiment, the activity or levelsof a Mam-IP are increased by administration of Mam, or derivative oranalog thereof, a nucleic acid encoding Mam, or an antibody thatimmunospecifically binds Mam or a fragment or derivative of the antibodycontains the binding domain thereof.

5.6.4 Origin of the Therapeutic

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, a humanMam:Mam-IP complex, or Mip30 or Mip6 protein, or derivative or analogthereof, nucleic acids encoding the members of the human Mam:Mam-IPcomplex, or human Mip30 or human Mip6, or a derivative or analogthereof, or an antibody to a human Mam:Mam-IP complex, or Mip30 or Mip6protein, or derivative thereof, is therapeutically or prophylacticallyadministered to a human patient.

5.6.5 Determination of the Effect of the Therapeutic

Preferably, suitable in vitro or in vivo assays are utilized todetermine the effect of a specific Therapeutic and whether itsadministration is indicated for treatment of the affected tissue.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells or cell types involved in a patient's disorder todetermine if a Therapeutic has a desired effect upon such cell types.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used. Additional descriptions and sources of Therapeutics thatcan be used according to the invention are found in Sections 5.1-5.3 and5.8 herein.

5.6.6 Neurodegenerative Disorders

Mam and certain binding partners of Mam (Notch) have been implicated inneurodegenerative disease. Within the developing mammalian nervoussystem, expression patterns of Notch homologs have been shown to beprominent in particular regions of the ventricular zone of the spinalcord, as well as in components of the peripheral nervous system, in anoverlapping but non-identical pattern. Notch expression in the nervoussystem appears to be limited to regions of cellular proliferation, andis absent from nearby populations of recently differentiated cells. Arat Notch ligand is also expressed within the developing spinal cord, indistinct bands of the ventricular zone that overlap with the expressiondomains of the Notch genes. The spatio-temporal expression pattern ofthis ligand correlates well with the patterns of cells committing tospinal cord neuronal fates, which demonstrates the usefulness of Notchas a marker of populations of cells for neuronal fates. Accordingly,Therapeutics of the invention, particularly but not limited to thosethat modulate (or supply) Mam:IP and complexes of Mam and Mam-IPs may beeffective in treating or preventing neurodegenerative disease.Therapeutics of the invention that modulate Mam:Mam-IP complexesinvolved in neurodegenerative disorders can be assayed by any methodknown in the art for efficacy in treating or preventing suchneurodegenerative diseases and disorders. Such assays include in vitroassays for regulated cell secretion, protein trafficking, and/or foldingor inhibition of apoptosis or in vivo assays using animal models ofneurodegenerative and/or developmental diseases or disorders, or any ofthe assays described in Sections 5.7.6 infra. Potentially effectiveTherapeutics, for example but not by way of limitation, promoteregulated cell maturation and prevent cell apoptosis in culture, orreduce neurodegeneration in animal models in comparison to controls.

Once a neurodegenerative disease or disorder has been shown to beamenable to treatment by modulation of Mam:Mam-IP complex activity, thatneurodegenerative disease or disorder can be treated or prevented byadministration of a Therapeutic that modulates Mam:Mam-IP complexformation (including supplying Mam:Mam-IP complexes).

Such diseases include all degenerative disorders involved with aging,especially osteoarthritis and neurodegenerative disorders.Neurodegenerative disorders that can be treated or prevented include butare not limited to those listed in Table I (see Isslebacher et al.,1997, In: Harrison's Principals of Internal Medicine, 13^(th) Ed.,McGraw Hill, New York). TABLE I NEURODEGENERATIVE DISORDERS Progressivedementia in the absence of other neurological signs Alzheimer's Disease(or early-onset AD) Senile dementia of the Alzheimer's type (or lateonset AD) Pick's Disease Syndromes combining progressive dementia withprominent neurological abnormalities Huntington's disease Multiplesystem atrophy (dementia combined with ataxia, Parkinson's disease,etc.) Progressive supranuclear palsy Diffuse Lewy body diseaseCorticodentatonigral degeneration Hallervorden-Spatz disease Progressivefamilial myoclonic epilepsy Syndromes of gradually developingabnormalities of posture and movement Parkinson's disease Striatonigraldegeneration Progressive supranuclear palsy Torsion dystonia Spasmodictorticollis and other restricted dyskinesias Familial tremor Gilles dela Tourette syndrome Syndromes of progressive ataxia Cerebellar corticaldegeneration Olivopontocerebellar atrophy Friedrich's ataxia and relatedspinocerebellar degenerations Shy-Drager syndrome Subacute necrotizingencephalopathy Motor neuron disease without sensory changes Amyotrophiclateral sclerosis Infantile spinal muscular atrophy Juvenile spinalmuscular atrophy Other forms of familial spinal muscular atrophy Primarylateral sclerosis Hereditary spastic paraplegia Motor neuron diseasewith sensory changes Peroneal muscular atrophy Hypertrophic interstitialpolyneuropathy Other forms of chronic progressive neuropathy Syndromesof progressive visual loss Retinitis pigmentosa

5.6.7 Oncogenesis

5.6.7.1 Malignancies

Components of Mam:Mam-IP complexes (i.e., Mam, Notch and Mip1 protein)have been implicated in regulation of cell proliferation. Accordingly,Therapeutics of the invention may be useful in treating or preventingdiseases or disorders associated with cell hyperproliferation or loss ofcontrol of cell proliferation, particularly cancers, malignancies andtumors. Therapeutics of the invention can be assayed by any method knownin the art for efficacy in treating or preventing malignancies andrelated disorders. Such assays include in vitro assays using transformedcells or cells derived from the tumor of a patient, or in vivo assaysusing animal models of cancer or malignancies, or any of the assaysdescribed in Sections 5.7 infra. Potentially effective Therapeutics, forexample but not by way of limitation, inhibit proliferation of tumors ortransformed cells in culture, or cause regression of tumors in animalmodels in comparison to controls, e.g., as described in Section 5.7,infra.

Accordingly, once a malignancy or cancer has been shown to be amenableto treatment by modulating (i.e., inhibiting, antagonizing, enhancing oragonizing) Mam:Mam-IP complex activity, or modulating Mip30 or Mip6,activity, that cancer or malignancy can be treated or prevented byadministration of a Therapeutic that modulates Mam:Mam-IP complexformation and function, or Mip30 or Mip6 function, including supplyingMam:Mam-IP complexes and the individual binding partners of a Mam:Mam-IPcomplex. Such cancers and malignancies include but are not limited tothose listed in Table II (for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia). TABLEII MALIGNANCIES AND RELATED DISORDERS Leukemia acute leukemia acutelymphocytic leukemia acute myelocytic leukemia myeloblastic-typepromyelocytic-type myelomonocytic-type monocytic-type erythroleukemiachronic leukemia chronic myelocytic (granulocytic) leukemia chroniclymphocytic leukemia Polycythemia vera Lymphoma Hodgkin's diseasenon-Hodgkin's disease Multiple myeloma Waldenström's macroglobulinemiaHeavy chain disease Solid tumors sarcomas and carcinomas fibrosarcomamyxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordomaangiosarcoma endotheliosarcoma lymphangiosarcomalymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumorleiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breastcancer ovarian cancer prostate cancer squamous cell carcinoma basal cellcarcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinomapapillary carcinoma papillary adenocarcinomas cystadenocarcinomamedullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatomabile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms'tumor cervical cancer uterine cancer testicular tumor lung carcinomasmall cell lung carcinoma bladder carcinoma epithelial carcinoma gliomaastrocytoma medulloblastoma craniopharyngioma ependymoma pinealomahemangioblastoma acoustic neuroma oligodendroglioma menangioma melanomaneuroblastoma retinoblastoma

In specific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders, aretreated or prevented in the bladder, breast, colon, lung, prostate,pancreas, or uterus.

5.6.7.2 Premalignant Conditions

The Therapeutics of the invention that are effective in treating canceror malignancies (e.g., as described above) can also be administered totreat premalignant conditions and to prevent progression to a neoplasticor malignant state, including but not limited to those disorders listedin Table II. Such prophylactic or therapeutic use is indicated inconditions known or suspected of preceding progression to neoplasia orcancer, in particular, where non-neoplastic cell growth consisting ofhyperplasia, metaplasia, or most particularly, dysplasia has occurred(for review of such abnormal growth conditions, see Robbins and Angell,1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.68-79). Hyperplasia is a form of controlled cell proliferation involvingan increase in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult cell or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. A typicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, skin, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a Therapeutic of theinvention that modulates Mam:Mam-IP complex activity, or that modulatesMip30 or Mip6 activity. As mentioned supra, such characteristics of atransformed phenotype include morphological changes, looser substratumattachment, loss of contact inhibition, loss of anchorage dependence,protease release, increased sugar transport, decreased serumrequirement, expression of fetal antigens, disappearance of the 250,000dalton cell surface protein, etc. (see also Id., pp. 84-90 forcharacteristics associated with a transformed or malignant phenotype).

In a specific embodiment, leukoplakia, a benign-appearing hyperplasticor dysplastic lesion of the epithelium, or Bowen's disease, a carcinomain situ, are pre-neoplastic lesions indicative of the desirability ofprophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammarydysplasia, particularly adenosis (benign epithelial hyperplasia)) isindicative of the desirability of prophylactic intervention.

In other embodiments, a patient that exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a Therapeutic: a chromosomaltranslocation associated with a malignancy (e.g., the Philadelphiachromosome for chronic myelogenous leukemia, t(14; 18) for follicularlymphoma, etc.), familial polyposis or Gardner's syndrome (possibleforerunners of colon cancer), benign monoclonal gammopathy (a possibleforerunner of multiple myeloma), and a first degree kinship with personshaving a cancer or precancerous disease showing a Mendelian (genetic)inheritance pattern (e.g., familial polyposis of the colon, Gardner'ssyndrome, hereditary exostosis, polyendocrine adenomatosis, medullarythyroid carcinoma with amyloid production and pheochromocytoma,Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen,retinoblastoma, carotid body tumor, cutaneous melanocarcinoma,intraocular melanocarcinoma, xeroderma pigmentosum, ataxiatelangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplasticanemia, and Bloom's syndrome; see Robbins and Angell, 1976, BasicPathology, 2nd Ed., W.B. Saunders Co., Philadelphia, pp. 112-113, etc.)

In another specific embodiment, a Therapeutic of the invention isadministered to a human patient to prevent progression to breast, colon,lung, pancreatic, prostate or uterine cancer, or melanoma or sarcoma.

5.6.7.3 Hyperproliferative and Dysproliferative Disorders

In another embodiment of the invention, a Therapeutic is administered totreat or prevent hyperproliferative or benign dysproliferativedisorders. Therapeutics of the invention can be assayed by any methodknown in the art for efficacy in treating or preventinghyperproliferative diseases or disorders, such assays include in vitrocell proliferation assays, in vitro or in vivo assays using animalmodels of hyperproliferative diseases or disorders, or any of the assaysdescribed in Section 5.7, infra. Potentially effective Therapeuticsinclude but are not limited to, Therapeutics that reduce cellproliferation in culture or inhibit growth or cell proliferation inanimal models in comparison to controls.

Accordingly, once a hyperproliferative disorder has been shown to beamenable to treatment by modulation of Mam:Mam-IP complex activity, orby modulation of Mip30 or Mip6 protein activity, that hyperproliferativedisease or disorder can be treated or prevented by administration of aTherapeutic that modulates Mam:Mam-IP complex formation, or thatmodulates Mip30 or Mip6 activity (including supplying a Mam:Mam-IPcomplex and/or the individual binding partners of a Mam:Mam-IP complex).

Specific embodiments are directed to treatment or prevention ofcirrhosis of the liver (a condition in which scarring has overtakennormal liver regeneration processes), treatment of keloid (hypertrophicscar) formation (disfiguring of the skin in which the scarring processinterferes with normal renewal), psoriasis (a common skin conditioncharacterized by excessive proliferation of the skin and delay in propercell fate determination), benign tumors, fibrocystic conditions, andtissue hypertrophy (e.g., prostatic hyperplasia).

5.6.8 Gene Therapy

In a specific embodiment, a nucleic acid molecule comprising a sequenceencoding Mam and/or a Mam-IP, or a Mip30 or Mip6 protein, or afunctional derivative thereof, are administered to modulate Mam:Mam-IPcomplexes, or to modulate Mip30 or Mip6 function, by way of genetherapy. In more specific embodiments, a nucleic acid or nucleic acidsencoding both Mam and a Mam-IP (e.g., Mip1, Mip30, Mip6), or functionalderivatives thereof, are administered by way of gene therapy. Genetherapy refers to therapy performed by the administration of a nucleicacid molecule to a subject. In this embodiment of the invention, thenucleic acid molecule produces its encoded protein(s) that mediates atherapeutic effect by modulating the Mam:Mam-IP complex, or bymodulating Mip30 or Mip6 function.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; and May, 1993, TIBTECH 11:155-215). Methodscommonly known in the art for recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY) and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the Therapeutic comprises a Mam and/or a Mam-IPnucleic acid, or a Mip30 or Mip6 nucleic acid, that is part of anexpression vector that expresses the Mam or Mam-IP protein(s), orexpresses a Mip30 or Mip6 protein, or fragment or a chimeric proteinthereof, in a suitable host. In particular, such a nucleic acid has apromoter(s) operably linked to the Mam and/or the Mam-IP codingregion(s), or linked to the Mip30 or Mip6 coding region, saidpromoter(s) being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the Mam and/or Mam-IP coding sequence, or theMip30 or Mip6 coding sequences, and any other desired sequences, areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intra-chromosomal expression ofthe Mam and the Mam-IP nucleic acids (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935, Zijlstra et al., 1989, Nature342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivoand ex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see, U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by encapsulation in liposomes, microparticles,or microcapsules, or by administering it in linkage to a peptide whichis known to enter the nucleus, or by administering it in linkage to aligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262:4429-4432), which can be used to target celltypes specifically expressing the receptors, etc. In another embodiment,a nucleic acid-ligand complex can be formed in which the ligandcomprises a fusogenic viral peptide that disrupts endosomes, preventinglysosomal degradation of the nucleic acid. In yet another embodiment,the nucleic acid can be targeted in vivo for cell specific uptake andexpression by targeting a specific receptor (see, e.g., InternationalPatent Publications WO 92/06180 by Wu et al., WO 92/22635 by Wilson etal., WO 92/20316 by Findeis et al., WO 93/14188 by Clarke et al., and WO93/20221 by Young). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad.Sci. USA 86:8932-8935, Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, a viral vector that contains the Mam and/orthe Mam-IP encoding nucleic acid sequence, or the Mip30 or Mip6 encodingnucleic acid sequence, is used. For example, a retroviral vector can beused (see Miller et al., 1993, Meth. Enzymol. 217:581-599). Theseretroviral vectors have been modified to delete retroviral sequencesthat are not necessary for packaging of the viral genome and integrationinto host cell DNA. The Mam and/or Mam-IP preferably both Mam andMam-IP) encoding nucleic acids, or Mip30 or Mip6 encoding nucleic acids,to be used in gene therapy is/are cloned into the vector, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy include: Clowes et al., 1994, J.Clin. Invest. 93:644-651, Kiem et al., 1994, Blood 83:1467-1473, Salmonsand Gunzberg, 1993, Human Gene Therapy 4:129-141, and Grossman andWilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10, demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434, Rosenfeld et al., 1992, Cell 68:143-155, andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).

Another approach to gene therapy involves transferring a gene into cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618, Cohen et al., 1993, Meth. Enzymol. 217:618-644,Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell, and isheritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes, and blood cells, such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, aMam and/or Mam-IP (preferably both Mam and Mam-IP) encoding nucleic acidmolecule, or a Mip30 or Mip6 encoding nucleic acid molecule, is/areintroduced into the cells such that the gene or genes are expressible bythe cells or their progeny, and the recombinant cells are thenadministered in vivo for therapeutic effect. In a specific embodiment,stem or progenitor cells are used. Any stem and/or progenitor cellswhich can be isolated and maintained in vitro can potentially be used inaccordance with this embodiment of the present invention. Such stemcells include but are not limited to hematopoietic stem cells (HSC),stem cells of epithelial tissues such as the skin and the lining of thegut, embryonic heart muscle cells, liver stem cells (InternationalPatent Publication WO 94/08598), and neural stem cells (Stemple andAnderson, 1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal laming Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,1980, Meth. Cell Bio. 21α:229; Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro ofHSCs can be used in this embodiment of the invention. Techniques bywhich this may be accomplished include (a) the isolation andestablishment of HSC cultures from bone marrow cells isolated from thefuture host, or a donor, or (b) the use of previously establishedlong-term HSC cultures, which may be allogeneic or xenogeneic.Non-autologous HSC are used preferably in conjunction with a method ofsuppressing transplantation immune reactions of the future host/patient.In a particular embodiment of the present invention, human bone marrowcells can be obtained from the posterior iliac crest by needleaspiration (see, e.g., Kodo et al., 1984, J. Clin. Invest.73:1377-1384). In a preferred embodiment of the present invention, theHSCs can be made highly enriched or in substantially pure form. Thisenrichment can be accomplished before, during, or after long-termculturing, and can be done by any technique known in the art. Long-termcultures of bone marrow cells can be established and maintained byusing, for example, modified Dexter cell culture techniques (Dexter etal., 1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques(Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods can be adapted for use to deliver a nucleic acidmolecule encoding the Mam and/or Mam-IP proteins, or functionalderivatives thereof, e.g., as described in Sections 5.1 and 5.2, supra.

5.6.9 Use of Antisense Oligonucleotides for Suppression of Mam:Mam-IPComplexes or for Suppression of Mip30 or Mip6 Protein Expression

In a specific embodiment, Mam:Mam-IP complex function or Mip30 or Mip6protein function is inhibited by use of antisense nucleic acids for Mamand/or a Mam-IP, (preferably both Mam and the Mam-IP), or individualantisense nucleic acids for Mip30 or Mip6. The present inventionprovides the therapeutic or prophylactic use of nucleic acids of atleast six nucleotides that are antisense to a gene or cDNA encoding Mamand/or a Mam-IP, or encoding Mip30 or Mip6, or a portion thereof. A Mamor a Mam-IP “antisense” nucleic acid as used herein refers to a nucleicacid capable of hybridizing to a portion of Mam or a Mam-IP nucleic acid(preferably mRNA) by virtue of some sequence complementarity. Theantisense nucleic acid may be complementary to a coding and/or noncodingregion of a Mam or Mam-IP mRNA. Such antisense nucleic acids haveutility as Therapeutics that inhibit Mam:Mam-IP complex formation oractivity, or Mip30 or Mip6 protein function or activity, and can be usedin the treatment or prevention of disorders as described, supra.

The antisense nucleic acids of the invention can be oligonucleotidesthat are double-stranded or single-stranded, RNA or DNA or amodification or derivative thereof, which can be directly administeredto a cell, or which can be produced intracellularly by transcription ofexogenous, introduced sequences.

In another embodiment, the invention is directed to methods forinhibiting the expression of Mam and/or a Mam-IP nucleotide sequence, orindividual Mip30 or Mip6 nucleotide sequences, in a prokaryotic oreukaryotic cell comprising providing the cell with an effective amountof a composition comprising an antisense nucleic acid of Mam and Mam-IP,or an antisense nucleic acid of Mip30 or Mip6, or a derivative thereof,of the invention.

The Mam and/or Mam-IP antisense nucleic acids are of at least sixnucleotides and are preferably oligonucleotides (ranging from 6 to about200 oligonucleotides). In specific aspects, the oligonucleotide is atleast about 10 nucleotides, at least about 15 nucleotides, at leastabout 100 nucleotides, or at least about 200 nucleotides. Theoligonucleotides can be DNA or RNA or chimeric mixtures or derivativesor modified versions thereof, single-stranded or double-stranded. Theoligonucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone. The oligonucleotide may include other appendinggroups such as peptides, or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556, Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652, PCT Publication No. WO 88/09810, published Dec. 15, 1988)transport across the blood-brain barrier (see, e.g., PCT Publication No.WO 89/10134, published Apr. 25, 1988), hybridization-triggered cleavageagents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976), orintercalation with other agents (see, e.g., Zon, 1988, Pharm. Res.5:539-549).

In a preferred aspect of the invention, a Mam and/or Mam-IP antisenseoligonucleotide is provided, preferably as single-stranded DNA. Theoligonucleotide may be modified at any position on its structure withconstituents generally known in the art.

The Mam and/or Mam-IP antisense oligonucleotides may comprise at leastone modified base moiety which is selected from the group including butnot limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5N-methoxycarboxymethyluracil, S-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, or a formacetal or analog thereof. In yet anotherembodiment, the oligonucleotide is a 2-anomeric oligonucleotide. Ananomeric oligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gautier et al., 1987, Nucl. Acids Res.15:6625-6641).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al., 1988, Nucl. Acids Res. 16:3209,methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

In a specific embodiment, the Mam and/or Mam-IP antisenseoligonucleotides comprise catalytic RNAs, or ribozymes (see, e.g., PCTInternational Publication WO 90/11364, published Oct. 4, 1990, Sarver etal., 1990, Science 247:1222-1225). In another embodiment, theoligonucleotide is a 2-0-methylribonucleotide (Inoue et al., 1987, Nucl.Acids Res. 15:6131-6148), or a chimeric RNA-DNA analog (Inoue et al.,1987, FEBS Lett. 215:327-330).

In an alternative embodiment, the Mam and/or Mam-IP antisense nucleicacids of the invention are produced intracellularly by transcriptionfrom an exogenous sequence. For example, a vector can be introduced invivo such that it is taken up by a cell, within which cell the vector ora portion thereof is transcribed, producing an antisense nucleic acid(RNA) of the invention. Such a vector would contain a sequence encodingMam and/or a Mam-IP (preferably, both a Mam and a Mam-IP antisensenucleic acid) antisense nucleic acid(s), or individual Mip30 or Mip6antisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art to be capable of replicationand expression in mammalian cells. Expression of the sequences encodingthe Mam and/or Mam-IP antisense RNAs can be by any promoter known in theart to act in mammalian, preferably human, cells. Such promoters can beinducible or constitutive. Such promoters include but are not limitedto: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a Mam or aMam-IP gene, preferably a human Mam or Mam-IP gene. However, absolutecomplementarity, although preferred, is not required. A sequence“complementary to at least a portion of an RNA,” as referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded Mam or Mam-IP antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches with aMam or Mam-IP RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

The Mam or Mam-IP antisense nucleic acid can be used to treat (orprevent) disorders of a cell type that expresses, or preferablyoverexpresses, the Mam:Mam-IP complex, or the Mip30 or Mip6 protein. Ina preferred embodiment, single-stranded DNA antisense Mam and Mam-IPoligonucleotides, or single-stranded DNA antisense to the same, orindividual Mip30 or Mip6 antisense oligonucleotides, or single-strandedDNA antisense to the same, is used.

Cell types that express or overexpress Mam and/or Mam-IP mRNA, or Mip30or Mip6 RNA can be identified by various methods known in the art. Suchmethods include, but are not limited to, hybridization with Mam- orMam-IP-specific nucleic acids (e.g., by Northern blot hybridization, dotblot hybridization, in situ hybridization), or by observing the abilityof RNA from the cell type to be translated in vitro into Mam or theMam-IP, e.g., by immunohistochemistry, ELISA, etc. In a preferredaspect, primary tissue from a patient can be assayed for Mam and/orMam-IP expression prior to treatment, e.g., by immunocytochemistry or insitu hybridization.

Pharmaceutical compositions of the invention (see Section 5.8, infra),comprising an effective amount of a Mam and/or a Mam-IP antisensenucleic acid in a pharmaceutically acceptable carrier, can beadministered to a patient having a disease or disorder that is of a typethat expresses or overexpresses Mam:Mam-IP complexes, Mam and/or Mam-IPmRNA, or Mip30 or Mip6 mRNA or protein.

The amount of Mam and/or Mam-IP antisense nucleic acid that will beeffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. Where possible, it is desirable todetermine the antisense cytotoxicity in vitro, and then in useful animalmodel systems prior to testing and use in humans.

In a specific embodiment, pharmaceutical compositions comprising Mam orMam-IP antisense nucleic acids are administered via liposomes,microparticles, or microcapsules. In various embodiments of theinvention, it may be useful to use such compositions to achievesustained release of the Mam and/or Mam-IP antisense nucleic acids. In aspecific embodiment, it may be desirable to utilize liposomes targetedvia antibodies to specific identifiable central nervous system celltypes (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:2448-2451, Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).

5.7 Assays of Mam:Mam-IP Complexes, and Mip30 or Mip6 Proteins

The functional activity of a Mam:Mam-IP complex, or the functionalactivity of a Mip30 or Mip6 protein, and derivatives, fragments andanalogs thereof, can be assayed by various methods known in the art.Potential modulators (e.g., inhibitors, agonists and antagonists) ofMam:Mam-IP complex activity, or of Mip30 or Mip6 activity (e.g.,anti-Mam:Mam-IP, anti-Mip30 or anti-Mip6 antibodies, and Mam or Mam-IPantisense nucleic acids) can be assayed for the ability to modulateMam:Mam-IP complex formation and/or activity, and for the ability tomodulate Mip30 or Mip6 activity.

5.7.1 Immunoassays

For example, in one embodiment, where one is assaying for the ability tobind or compete with wild-type Mam:Mam-IP complexes, or Mip30 or Mip6protein, for binding to anti-Mam:Mam-IP antibodies, or anti-Mip30 oranti-Mip6 antibodies, various immunoassays known in the art can be used,including but not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), Western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays,immunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

5.7.2 Assays for Gene Expression

The expression of the Mam and/or Mam-IP genes (both endogenous genes andthose expressed from cloned DNA containing these genes) can be detectedusing techniques known in the art, including but not limited to Southernhybridization (Southern, 1975, J. Mol. Biol. 98: 503-517), Northernhybridization (e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. USA80: 4094-4098), restriction endonuclease mapping (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., Cold Spring HarborLaboratory Press, New York), DNA sequence analysis, polymerase chainreaction amplification (PCR, U.S. Pat. Nos. 4,683,202, 4,683,195, and4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. USA85:7652-7657; Ochman et al., 1988, Genetics 120:621-623; and Loh et al.,1989, Science 243:217-220), or RNase protection (Current Protocols inMolecular Biology, John Wiley and Sons, New York, 1997) with probesspecific for Mam or Mam-IP genes, in various cell types. Methods ofamplification other than PCR commonly known in the art can be employed.In one embodiment, Southern hybridization can be used to detect geneticlinkage of Mam or Mam-IP gene mutations to physiological or pathologicalstates. Various cell types, at various stages of development, can becharacterized for their expression of Mam and/or a Mam-IP (particularlyexpression of Mam and/or a Mam-IP at the same time and in the samecells), or Mip30 or Mip6 protein expression. The stringency of thehybridization conditions for northern or Southern blot analysis can bemanipulated to ensure detection of nucleic acids with the desired degreeof relatedness to the specific probes used. Modifications to thesemethods and other methods commonly known in the art can be used.

5.7.3 Binding Assays

Derivatives (e.g., fragments) and analogs of Mam-IPs can be assayed forbinding to Mam by any method known in the art, for example the modifiedyeast two hybrid assay system described in Section 6, infra,immunoprecipitation with an antibody that binds to Mam in a complexfollowed by analysis by size fractionation of the immunoprecipitatedproteins (e.g., by denaturing or nondenaturing polyacrylamide gelelectrophoresis), Western analysis, non-denaturing gel electrophoresis,etc.

5.7.4 Assays for Biological Activity

One embodiment of the invention provides a method for screening aderivative or analog of Mam for biological activity comprisingcontacting said derivative or analog of Mam with a protein selected fromthe group consisting of Mip1, Mip30 and Mip6, and detecting theformation of a complex between said derivative or analog of Mam and saidprotein; wherein detecting formation of said complex indicates that saidderivative or analog of Mam has biological (e.g., binding) activity.Additionally, another embodiment of the invention relates to a methodfor screening a derivative or analog of a protein selected from thegroup consisting of Mip1, Mip30 and Mip6 for biological activitycomprising contacting said derivative or analog of said protein withMam; and detecting the formation of a complex between said derivative oranalog of said protein and Mam; wherein detecting the formation of saidcomplex indicates that said derivative or analog of said protein hasbiological activity.

5.7.5 Methods of Modulating the Protein Activity

The present invention also provides methods of modulating the activityof a protein that can participate in a Mam:Mam-IP complex (e.g., Mam,Mip1, Mip30, or Mip6) by administration of a binding partner of thatprotein, or derivative or analog thereof. Mam and derivatives andanalogs thereof, can be assayed for the ability to modulate the activityor level of a Mam-IP by contacting a cell, or administering to ananimal, expressing a Mam-IP gene with a Mam protein, or a nucleic acidencoding a Mam protein, or an antibody that immunospecifically binds theMam protein, or a fragment or derivative of said antibody containing thebinding domain thereof, and measuring a change in Mam-IP levels oractivity, wherein a change in Mam-IP levels or activity indicates thatMam can modulate Mam-IP levels or activity. Alternatively, a Mam-IP canbe assayed for the ability to modulate the activity or levels of a Mamprotein by contacting a cell, or administering to an animal, expressinga Mam gene with a Mam-IP, or a nucleic acid encoding a Mam-IP, or anantibody that immunospecifically binds to a Mam-IP, or a fragment orderivative of said antibody containing the binding domain thereof,wherein a change in Mam levels or activity indicates that the Mam-IP canmodulate Mam levels or activity.

The Mam:Mam-IP complex, or Mip30 or Mip6 protein, or derivative, analog,or fragment thereof, can also be screened for activity in modulating theactivity of Mam and the Mam binding partners particularly Mip1, Mip30and Mip6 (i.e., the Mam-IPs, involved in particular Mam:Mam-IPcomplexes). The complexes and proteins of the invention can be screenedfor the ability to modulate (i.e., increase or decrease) Mam:Mam-IPcomplexes, as specified below.

Mip30 contains seven C2H2 zinc-finger repeat domains, a HMG-1 and HMG-YDNA-binding domain (A+T-hook), and a bipartite nuclear localizationsignal. Mip6 contains a bipartite nuclear localization signal.

5.7.6 Assays for Treatment of Neurodegeneration Disorders

The Mam:Mam-IP complexes particularly the Mam:Mip1, Mam:Mip30 andMam:Mip6 complexes), derivatives, analogs and fragments thereof, nucleicacids encoding the Mam and Mam-IP genes, anti-Mam:Mam-IP antibodies, andother modulators of Mam:Mam-IP complex activity, can be tested foractivity in treating or preventing neurodegenerative disease in in vitroand in vivo assays.

In one embodiment, a Therapeutic of the invention can be assayed foractivity in treating or preventing neurodegenerative disease bycontacting cultured cells that exhibit an indicator of aneurodegenerative disease, such as overexpression of the β-A4 peptide,in vitro with the Therapeutic, and comparing the level of said indicatorin the cells contacted with the Therapeutic with said level of saidindicator in cells not so contacted, wherein a lower level in saidcontacted cells indicates that the Therapeutic has activity in treatingor preventing neurodegenerative disease. Specific examples of cellculture models for neurodegenerative disease include, but are notlimited to, cultured rat endothelial cells from affected and nonaffectedindividuals (Maneiro et al., 1997, Methods Find. Exp. Clin. Pharmacol.19:5-12), P19 murine embryonal carcinoma cells (Hung et al., 1992, Proc.Natl. Acad. Sci. USA 89:9439-9443), and dissociated cell cultures ofcholinergic neurons from the nucleus basalis of Meynert (Nakajima etal., 1985, Proc. Natl. Acad. Sci. USA, 82:6325-6329).

In another embodiment, a Therapeutic of the invention can be assayed foractivity in treating or preventing neurodegenerative disease byadministering the Therapeutic to a test animal that exhibits symptoms ofa neurodegenerative disease, such as premature development of cognitivedeficiencies in transgenic animals expressing β-APP, or that ispredisposed to develop symptoms of a neurodegenerative disease; andmeasuring the change in said symptoms of the neurodegenerative diseaseafter administration of said Therapeutic, wherein a reduction in theseverity of the symptoms of the neurodegenerative disease or preventionof the symptoms of the neurodegenerative disease, indicates that theTherapeutic has activity in treating or preventing neurodegenerativedisease. Such a test animal can be any one of a number of animal modelsknown in the art for neurodegenerative disease. These models, includingthose for Alzheimer's Disease and mental retardation of trisomy 21,which accurately mimic the natural human neurodegenerative disease(Campbell, et al., 1997, Mol. Psychiatry 2:125-129; Schultz et al.,1997, Mol. Cell. Biochem. 174:193-197; Oron et al., 1997, J. Neural.Transm. Suppl. 49:77-84). Examples of specific models, include but arenot limited to, the partial trisomy 16 mouse (Holtzman et al., 1996,Proc. Natl. Acad. Sci. USA 93:13333-13338), bilateral nucleus basalismagnocellularis-lesioned rats (Popovic et al., 1996, Int. J. Neurosci.86:281-299), the aged rat (Muir, 1997, Pharmacol. Biochem. Behav.56:687-696), the PDAPP transgenic mouse model of Alzheimer's disease(Johnson-Wood et al., 1997, Proc. Natl. Acad. Sci. USA 94:1550-1555),and experimental autoimmune dementia (Oron et al., 1997, J. NeuralTransm. Suppl. 49:77-84).

5.7.7 Assays for Treatment of Tumorigenesis

Mam and several of the identified binding partners of Mam (e.g., Mip1)have roles in the control of mitosis and cell proliferation and,therefore, cell-transformation and tumorigenesis. Accordingly, methodsof the invention are provided for screening Mam:Mam-IP complexes,proteins, and fragments, derivatives and analogs of the foregoing, foractivity in altering cell proliferation, cell transformation and/ortumorigenesis in vitro and in vivo.

The Mam:Mam-IP complexes or Mip30 or Mip6 proteins, derivatives,fragments, and analogs thereof, can be assayed for activity to alter(i.e., increase or decrease) cell proliferation in cultured cells invitro using methods which are well known in the art for measuring cellproliferation. Specific examples of cell culture models include, but arenot limited to, for lung cancer, primary rat lung tumor cells (Swaffordet al., 1997, Mol. Cell. Biol., 17:1366-1374) and large-cellundifferentiated cancer cell lines (Mabry et al., 1991, Cancer Cells,3:53-58), colorectal cell lines for colon cancer (Park and Gazdar, 1996,J. Cell Biochem. Suppl. 24:131-141), multiple established cell lines forbreast cancer (Hambly et al., 1997, Breast Cancer Res. Treat.43:247-258; Gierthy et al., 1997, Chemosphere 34:1495-1505; Prasad andChurch, 1997, Biochem. Biophys. Res. Commun. 232:14-19), a number ofwell-characterized cell models for prostate cancer (Webber et al., 1996,Prostate, Part 1, 29:386-394; Part 2, 30:58-64; and Part 3, 30:136-142;Boulikas, 1997, Anticancer Res. 17:1471-1505), for genitourinarycancers, continuous human bladder cancer cell lines (Ribeiro et al.,1997, Int. J. Radiat. Biol. 72:11-20), organ cultures of transitionalcell carcinomas (Booth et al., 1997, Lab Invest. 76:843-857), and ratprogression models (Vet et al., 1997, Biochim. Biophys Acta 1360:39-44),and established cell lines for leukemias and lymphomas (Drexler, 1994,Leuk. Res. 18:919-927, Tohyama, 1997, Int. J. Hematol. 65:309-317).

For example, but not by way of limitation, cell proliferation can beassayed by measuring ³H-thymidine incorporation, by direct cell count,by detecting changes in transcriptional activity of known genes, such asproto-oncogenes (e.g., c-fos and c-myc), by detecting changes in cellcycle markers, etc. Accordingly, one embodiment of the present inventionprovides a method of screening Mam:Mam-IP complexes, or Mip30 or Mip6protein, and fragments, derivatives, and analogs thereof, for activityin altering (i.e., increasing or decreasing) proliferation of cells invitro, comprising contacting the cells with a Mam:Mam-IP complex, or aMip30 or Mip6 protein, or a derivative, analog, or fragment thereof,measuring the proliferation of cells that have been so contacted, andcomparing the proliferation of the cells so contacted with a complex orprotein of the invention with the proliferation of cells not socontacted with the complex or protein of the invention, wherein in achange in the level of proliferation in said contacted cells indicatesthat the complex or protein of the invention has activity to alter cellproliferation.

The Mam:Mam-IP complexes, or Mip30 or Mip6 protein, derivative, fragmentor analog thereof, can also be screened for activity in inducing orinhibiting cell transformation (or progression to malignant phenotype)in vitro. The complexes and proteins of the invention can be screened bycontacting either cells with a normal phenotype (for assaying for celltransformation) or a transformed cell phenotype (for assaying forinhibition of cell transformation) with the complex or protein of theinvention, and examining the cells for acquisition or loss ofcharacteristics associated with a transformed phenotype (a set of invitro characteristics associated with a tumorigenic ability in vivo),for example, but not limited to, colony formation in soft agar, a morerounded cell morphology, looser substratum attachment, loss of contactinhibition, loss of anchorage dependence, release of proteases such asplasminogen activator, increased sugar transport, decreased serumrequirement, expression of fetal antigens, disappearance of the 250 kDsurface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed.,John Wiley & Sons, New York, pp. 436-446).

The Mam:Mam-IP complexes, or Mip30 or Mip6 protein, derivative,fragment, or analog thereof, can also be screened for activity topromote or inhibit tumor formation in vivo in a non-human test animal. Avast number of animal models of hyperproliferative disorders, includingtumorigenesis and metastatic spread, are known in the art (see Table317-1, Chapter 317, “Principals of Neoplasia,” in Harrison's Principalsof Internal Medicine, 13th Edition, Isselbacher et al., eds.,McGraw-Hill, New York, p. 1814, and Lovejoy et al., 1997, J. Pathol.181:130-135). Specific examples include for lung cancer, transplantationof tumor nodules into rats (Wang et al., 1997, Ann. Thorac. Surg.64:216-219) or establishment of lung cancer metastases in SCID micedepleted of NK cells (Yono and Sone, 1997, Gan To Kagaku Ryoho24:489-494); for colon cancer, colon cancer transplantation of humancolon cancer cells into nude mice (Gutman and Fidler, 1995, World J.Surg. 19:226-234), the cotton top tamarin model of human ulcerativecolitis (Warren, 1996, Aliment. Pharmacol. Ther. 10 Supp 12:45-47) andmouse models with mutations of the adenomatous polyposis tumorsuppressor (Polakis, 1997, Biochim. Biophys. Acta 1332:F127-F147); forbreast cancer, transgenic models of breast cancer (Dankort and Muller,1996, Cancer Treat. Res. 83:71-88; Amundadittir et al., 1996, BreastCancer Res. Treat. 39:119-135) and chemical induction of tumors in rats(Russo and Russo, 1996, Breast Cancer Res. Treat. 39:7-20); for prostatecancer, chemically-induced and transgenic rodent models, and humanxenograft models (Royai et al., 1996, Semin. Oncol. 23:35-40); forgenitourinary cancers, induced bladder neoplasm in rats and mice (Oyasu,1995, Food Chem. Toxicol 33:747-755) and xenografts of humantransitional cell carcinomas into nude rats (Jarrett et al., 1995, J.Endourol. 9:1-7); and for hematopoietic cancers, transplanted allogeneicmarrow in animals (Appelbaum, 1997, Leukemia 11 (Suppl. 4):S15-S17).Further, general animal models applicable to many types of cancer havebeen described, including, but not restricted to, the p53-deficientmouse model (Donehower, 1996, Semin. Cancer Biol. 7:269-278), the Minmouse (Shoemaker et al., 1997, Biochem. Biophys. Acta, 1332:F25-F48),and immune responses to tumors in rat (Frey, 1997, Methods, 12:173-188).

For example, the complexes and proteins of the present invention can beadministered to non-human test animals (preferably test animalspredisposed to develop a type of tumor) and the non-human test animalsubsequently examined for an increased incidence of tumor formation incomparison with controls not administered the complex or protein of theinvention. Alternatively, the complexes and proteins of the presentinvention can be administered to non-human test animals having tumors(e.g., animals in which tumors have been induced by introduction ofmalignant, neoplastic, or transformed cells, or by administration of acarcinogen) and subsequently examining the tumors in the test animalsfor tumor regression in comparison to controls not administered thecomplex a protein of the present invention.

In one embodiment of the present invention, a molecule that modulatesactivity of Mam or a protein selected from the group consisting of Mip1,Mip30 and Mip6, or a complex of Mam and said protein, is identified bycontacting one or more candidate molecules with Mam in the presence ofsaid protein; and measuring the amount of complex that forms between Mamand said protein; wherein an increase or decrease in the amount ofcomplex that forms relative to the amount that forms in the absence ofthe candidate molecules indicates that the molecules modulate theactivity of Mam or said protein or said complex of Mam and said protein.In preferred embodiments, modulators are identified by administering acandidate molecule to a transgenic non-human animal expressing both Mamand a Mam-IP from promoters that are not the native Mam or the nativeMam-IP promoters, more preferably where the candidate molecule is alsorecombinantly expressed in the transgenic non-human animal.Alternatively, the method for identifying such modulators can be carriedout in vitro, preferably with purified Mam, purified Mam-IP, and apurified candidate molecule.

Methods that can be used to carry out the foregoing are commonly knownin the art. Agents to be screened can be provided as mixtures of alimited number of specified compounds, or as compound libraries, peptidelibraries and the like. Agents to be screened may also include all formsof antisera, antisense nucleic acids, etc., that can modulate Mam:Mam-IPcomplex activity, or modulate a Mip30 or Mip6 activity.

Exemplary libraries of candidate molecules are described in Section5.4.1, supra.

In a specific embodiment, screening can be carried out by contacting thelibrary members with a Mam:Mam-IP complex, or with a Mip30 or Mip6protein (or encoding nucleic acid molecule or derivative) immobilized ona solid phase, and harvesting those library members that bind to theprotein (or nucleic acid or derivative). Examples of such screeningmethods, termed “panning” techniques, are described by way of example inParmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992,BioTechniques 13:422-427; International Patent Publication No. WO94/18318; and in references cited hereinabove.

In a specific embodiment, fragments and/or analogs of Mam or a Mam-IP,especially peptidomimetics, are screened for activity as competitive ornon-competitive inhibitors of Mam:Mam-IP complex formation, and therebyinhibit Mam:Mam-IP complex activity.

In a preferred embodiment, molecules that bind to a Mam:Mam-IP complex,or to a Mip30 or Mip6 protein, can be screened for by using the modifiedyeast two hybrid system described in Section 5.8.1 infra, andexemplified in Section 6.1, infra.

In one embodiment, agents that modulate (i.e., inhibit, antagonize oragonize) Mam:Mam-IP complex activity can be screened for using a bindinginhibition assay, wherein agents are screened for their ability toinhibit formation of a Mam:Mam-IP complex under aqueous, orphysiological, binding conditions in which Mam:Mam-IP complex formationoccurs in the absence of the agent to be tested. Agents that interferewith the formation of Mam:Mam-IP complexes are identified as antagonistsof complex formation. Agents that eliminate the formation of Mam:Mam-IPcomplexes are identified as inhibitors of complex formation. Agents thatenhance the formation of Mam:Mam-IP complexes are identified as agonistsof complex formation.

Methods for screening may involve labeling the complex proteins withradioligands (e.g., ¹²⁵I, or ³H), magnetic ligands (e.g., paramagneticbeads covalently attached to photobiotin acetate), fluorescent ligands(e.g., fluorescein or rhodamine) or enzyme ligands (e.g., luciferase orbeta-galactosidase). The reactants that bind in solution can then beisolated by one of many techniques known in the art, including but notrestricted to, co-immunoprecipitation of the labeled moiety usingantisera against the unlabeled binding partner (or a binding partnerlabeled with a distinguishable marker from that used on the labeledmoiety), immunoaffinity chromatography, size exclusion chromatography,and gradient density centrifugation. In a preferred embodiment, onebinding partner is a small fragment or peptidomimetic that is notretained by a commercially available filter. Upon binding, the labeledspecies is then unable to pass through the filter, providing for asimple assay of complex formation.

Methods commonly known in the art are used to label at least one of themembers of the Mam:Mam-IP complex. Suitable labeling includes, but isnot limited to, radiolabeling by incorporation of radiolabeled aminoacids, e.g., ³H-leucine or ³⁵S-methionine, radiolabeling bypost-translational iodination with ¹²⁵I or ¹³¹I using the chloramine Tmethod, Bolton-Hunter reagents, etc., labeling with ³²P using a kinaseand inorganic radiolabeled phosphorous, biotin labeling withphotobiotin-acetate and sunlamp exposure, etc. In cases where one of themembers of the Mam:Mam-IP complex is immobilized, e.g., as describedinfra, the free species is labeled. Where neither of the interactingspecies is immobilized, each can be labeled with a distinguishablemarker such that isolation of both moieties can be followed to providefor more accurate quantitation, and to distinguish the formation ofhomomeric from heteromeric complexes. Methods that utilize accessoryproteins that bind to one of the modified interactants to improve thesensitivity of detection, increase the stability of the complex, etc.are provided.

Typical binding conditions are, for example, but not by way oflimitation, in an aqueous salt solution of 10-250 mM NaCl, 5-50 mMTris-HCl, pH 5-8, and 0.5% Triton X-100 or other detergent that improvesthe specificity of interaction. Metal chelators and/or divalent cationsmay be added to improve binding and/or reduce proteolysis. Reactiontemperatures may include 4, 10, 15, 22, 25, 35, or 42 degrees Celsius,and time of incubation is typically at least 15 seconds, but longertimes are preferred to allow binding equilibrium to occur. ParticularMam:Mam-IP complexes can be assayed using routine protein binding assaysto determine optimal binding conditions for reproducible binding.

The physical parameters of complex formation can be analyzed byquantitation of complex formation using assay methods specific for thelabel used, e.g., liquid scintillation spectroscopy for radioactivitydetection, enzyme activity measurements for enzyme labeling, etc. Thereaction results are then analyzed utilizing Scatchard analysis, Hillanalysis, and other methods commonly known in the art (see, e.g.,Proteins, Structures, and Molecular Principles, 2^(nd) Edition (1993)Creighton, Ed., W.H. Freeman and Company, New York).

In a second common approach to binding assays, one of the bindingspecies is immobilized on a filter, in a microtiter plate well, in atest tube, to a chromatography matrix, etc., either covalently ornon-covalently. Proteins can be covalently immobilized using any methodwell known in the art, for example, but not limited to the method ofKadonaga and Tjian (1986, Proc. Natl. Acad. Sci. USA 83:5889-5893,1986), i.e., linkage to a cyanogen-bromide derivatized substrate such asCNBr-Sepahrose 4B. Where needed, the use of spacers can reduce sterichindrance by the substrate. Non-covalent attachment of proteins to asubstrate include, but are not limited to, attachment of a protein to acharged surface, binding with specific antibodies, binding to a thirdunrelated interacting protein.

In one embodiment, immobilized Mam is used to assay for binding with aradioactively-labeled Mam-IP in the presence and absence of a compoundto be tested for its ability to modulate Mam:Mam-IP complex formation.The binding partners are allowed to bind under aqueous, orphysiological, conditions (e.g., the conditions under which the originalinteraction was detected). Conversely, in another embodiment, the Mam-IPis immobilized and contacted with the labeled Mam protein or derivativethereof under binding conditions.

Assays of agents (including cell extracts or library pools) forcompetition for binding of one member of a Mam:Mam-IP complex (orderivatives thereof) with the other member of the Mam:Mam-IP complex(labeled by any means, e.g., those means described supra), are providedto screen for competitors of Mam:Mam-IP complex formation.

In specific embodiments, blocking agents to inhibit non-specific bindingof reagents to other protein components, or absorptive losses ofreagents to plastics, immobilization matrices, etc., are included in theassay mixture. Blocking agents include, but are not restricted to,bovine serum albumin, beta-casein, nonfat dried milk, Denhardt'sreagent, Ficoll, polyvinylpyrolidine, nonionic detergents (NP40, TritonX-100, Tween 20, Tween 80, etc.), ionic detergents (e.g., SDS, LDS,etc.), polyethylene glycol, etc. Appropriate blocking agentconcentrations are utilized to allow Mam:Mam-IP complex formation.

After binding is performed, unbound, labeled protein is removed with thesupernatant, and the immobilized protein with any bound, labeled proteinis washed extensively. The amount of label bound is then quantitatedusing standard methods known in the art to detect the label.

5.8.1 Assays for Proteins-Protein Interactions

One aspect of the present invention provides methods for assaying andscreening fragments, derivatives and analogs of Mam interacting proteins(for binding to a Mam peptide). Derivatives, analogs and fragments ofMam-IPs that interact with Mam can be identified by means of a yeast twohybrid assay system (Fields and Song, 1989, Nature 340:245-246 and U.S.Pat. No. 5,283,173). Because the interactions are screened for in yeast,the intermolecular protein interactions detected in this system occurunder physiological conditions that mimic the conditions in mammaliancells (Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9581).

Identification of interacting proteins by the improved yeast two hybridsystem is based upon the detection of expression of a reporter gene, thetranscription of which is dependent upon the reconstitution of atranscriptional regulator by the interaction of two proteins, each fusedto one half of the transcriptional regulator. The “bait” (Mam orderivative or analog) and “prey” (proteins to be tested for ability tointeract with the bait) proteins are expressed as fusion proteins to aDNA binding domain, and to a transcriptional regulatory domain,respectively, or vice versa. In various specific embodiments, the preyhas a complexity of at least about 50, about 100, about 500, about1,000, about 5,000, about 10,000, or about 50,000; or has a complexityin the range of about 25 to about 100,000, about 100 to about 100,000,about 50,000 to about 100,000, or about 100,000 to about 500,000. Forexample, the prey population can be one or more nucleic acids encodingmutants of a Mam-IP (e.g., as generated by site-directed mutagenesis oranother method of making mutations in a nucleotide sequence).Preferably, the prey populations are proteins encoded by DNA, e.g., cDNAor genomic DNA or synthetically generated DNA. For example, thepopulations can be expressed from chimeric genes comprising cDNAsequences from an un-characterized sample of a population of cDNA frommammalian RNA.

In a specific embodiment, recombinant biological libraries expressingrandom peptides can be used as the source of prey nucleic acids.

In another embodiment, the invention provides methods of screening forinhibitors or enhancers of the protein interactants identified herein.Briefly, the protein-protein interaction assay can be carried out asdescribed herein, except that it is done in the presence of one or morecandidate molecules. An increase or decrease in reporter gene activityrelative to that present when the one or more candidate molecules areabsent indicates that the candidate molecule has an effect on theinteracting pair. In a preferred method, inhibition of the interactionis selected for (i.e., inhibition of the interaction is necessary forthe cells to survive), for example, where the interaction activates theURA3 gene, causing yeast to die in medium containing the chemical5-fluoroorotic acid (Rothstein, 1983, Meth. Enzymol. 101: 167-180). Theidentification of inhibitors of such interactions can also beaccomplished, for example, but not by way of limitation, usingcompetitive inhibitor assays, as described supra.

In general, proteins of the bait and prey populations are provided asfusion (chimeric) proteins (preferably by recombinant expression of achimeric coding sequence) comprising each protein contiguous to apre-selected sequence. For one population, the pre-selected sequence isa DNA binding domain. The DNA binding domain can be any DNA bindingdomain, as long as it specifically recognizes a DNA sequence within apromoter. For example, the DNA binding domain is of a transcriptionalactivator or inhibitor. For the other population, the pre-selectedsequence is an activator or inhibitor domain of a transcriptionalactivator or inhibitor, respectively. The regulatory domain alone (notas a fusion to a protein sequence) and the DNA-binding domain alone (notas a fusion to a protein sequence) preferably do not detectably interact(so as to avoid false positives in the assay). The assay system furtherincludes a reporter gene operably linked to a promoter that contains abinding site for the DNA binding domain of the transcriptional activator(or inhibitor). Accordingly, in the present method of the presentinvention, binding of a Mam fusion protein to a prey fusion proteinleads to reconstitution of a transcriptional activator (or inhibitor)which activates (or inhibits) expression of the reporter gene. Theactivation (or inhibition) of transcription of the reporter gene occursintracellularly, e.g., in prokaryotic or eukaryotic cells, preferably incell culture.

The promoter that is operably linked to the reporter gene nucleotidesequence can be a native or non-native promoter of the nucleotidesequence, and the DNA binding site(s) that are recognized by the DNAbinding domain portion of the fusion protein can be native to thepromoter (if the promoter normally contains such binding site(s)) ornon-native to the promoter. Thus, for example, one or more tandem copies(e.g., 4 or 5 copies) of the appropriate DNA binding site can beintroduced upstream of the TATA box in the desired promoter (e.g. in thearea of about position −100 to about −400). In a preferred aspect, 4 or5 tandem copies of the 17 bp UAS (GAL4 DNA binding site) are introducedupstream of the TATA box in the desired promoter, which is upstream ofthe desired coding sequence for a selectable or detectable marker. In apreferred embodiment, the GAL1-10 promoter is operably fused to thedesired nucleotide sequence; the GAL1-10 promoter already contains 5binding sites for GAL4.

Alternatively, the transcriptional activation binding site of thedesired gene(s) can be deleted and replaced with GAL4 binding sites(Bartel et al., 1993, BioTechniques 14:920-924, Chasman et al., 1989,Mol. Cell. Biol. 9:4746-4749). The reporter gene preferably contains thesequence encoding a detectable or selectable marker, the expression ofwhich is regulated by the transcriptional activator, such that themarker is either turned on or off in the cell in response to thepresence of a specific interaction. Preferably, the assay is carried outin the absence of background levels of the transcriptional activator(e.g., in a cell that is mutant or otherwise lacking in thetranscriptional activator). In one embodiment, more than one reportergene is used to detect transcriptional activation, e.g., one reportergene encoding a detectable marker and one or more reporter genesencoding different selectable markers. The detectable marker can be anymolecule that can give rise to a detectable signal, e.g., a fluorescentprotein or a protein that can be readily visualized or that isrecognizable by a specific antibody. The selectable marker can be anyprotein molecule that confers the ability to grow under conditions thatdo not support the growth of cells not expressing the selectable marker,e.g., the selectable marker is an enzyme that provides an essentialnutrient and the cell in which the interaction assay occurs is deficientin the enzyme and the selection medium lacks such nutrient. The reportergene can either be under the control of the native promoter thatnaturally contains a binding site for the DNA binding protein, or underthe control of a heterologous or synthetic promoter.

The activation domain and DNA binding domain used in the assay can befrom a wide variety of transcriptional activator proteins, as long asthese transcriptional activators have separable binding andtranscriptional activation domains. For example, the GAL4 protein of S.cerevisiae (Ma et al., 1987, Cell 48:847-853), the GCN4 protein of S.cerevisiae (Hope and Struhl, 1986, Cell 46:885-894), the ARD1 protein ofS. cerevisiae (Thukral et al., 1989, Mol. Cell. Biol. 9:2360-2369), andthe human estrogen receptor (Kumar et al., 1987, Cell 51:941-951), haveseparable DNA binding and activation domains. The DNA binding domain andactivation domain that are employed in the fusion proteins need not befrom the same transcriptional activator. In a specific embodiment, aGAL4 or LEXA DNA binding domain is employed. In another specificembodiment, a GAL4 or herpes simplex virus VP16 (Triezenberg et al.,1988, Genes Dev. 2:730-742) activation domain is employed. In a specificembodiment, amino acids 1-147 of GAL4 (Ma et al., 1987, Cell 48:847-853;Ptashne et al., 1990, Nature 346:329-331) is the DNA binding domain, andamino acids 411-455 of VP16 (Triezenberg et al., 1988, Genes Dev.2:730-742; Cress et al., 1991, Science 251:87-90) comprise theactivation domain.

In a preferred embodiment, the yeast transcription factor GAL4 isreconstituted by protein-protein interaction and the host strain ismutant for GAL4. In another embodiment, the DNA-binding domain is Ace1Nand/or the activation domain is Ace1, the DNA binding and activationdomains of the Ace1 protein, respectively. Ace1 is a yeast protein thatactivates transcription from the CUP1 operon in the presence of divalentcopper. CUP1 encodes metallothionein, which chelates copper, and theexpression of CUP1 protein allows growth in the presence of copper,which is otherwise toxic to the host cells. The reporter gene can alsobe a CUP1-lacZ fusion that expresses the enzyme beta-galactosidase(detectable by routine chromogenic assay) upon binding of areconstituted Ace1N transcriptional activator (see Chaudhuri et al.,1995, FEBS Letters 357:221-226). In another specific embodiment, the DNAbinding domain of the human estrogen receptor is used, with a reportergene driven by one or three estrogen receptor response elements (LeDouarin et al., 1995, Nucl. Acids. Res. 23:876-878).

The DNA binding domain and the transcriptional activator/inhibitordomain each preferably has a nuclear localization signal (see Ylikomi etal., 1992, EMBO J. 11:3681-3694, Dingwall and Laskey, 1991, TIBS16:479-481) functional in the cell in which the fusion proteins are tobe expressed.

To facilitate isolation of the encoded proteins, the fusion constructscan further contain sequences encoding affinity tags such asglutathione-5-transferase or maltose-binding protein or an epitope of anavailable antibody, for affinity purification (e.g., binding toglutathione, maltose, or a particular antibody specific for the epitope,respectively) (Allen et al., 1995, TIBS 20:511-516). In anotherembodiment, the fusion constructs further comprise bacterial promotersequences for recombinant production of the fusion protein in bacterialcells.

The host cell in which the interaction assay occurs can be any cell,prokaryotic or eukaryotic, in which transcription of the reporter genecan occur and be detected, including, but not limited to, mammalian(e.g., monkey, mouse, rat, human, bovine), chicken, bacterial, or insectcells, and is preferably a yeast cell. Expression constructs encodingand capable of expressing the binding domain fusion proteins, thetranscriptional activation domain fusion proteins, and the reporter geneproduct(s) are provided within the host cell, by mating of cellscontaining the expression constructs, or by cell fusion, transformation,electroporation, microinjection, etc. In a specific embodiment in whichthe assay is carried out in mammalian cells (e.g., hamster cells), theDNA binding domain is the GAL4 DNA binding domain, the activation domainis the herpes simplex virus VP16 transcriptional activation domain, andthe reporter gene contains the desired coding sequence operably linkedto a minimal promoter element from the adenovirus E1B gene driven byseveral GAL4 DNA binding sites (see Fearon et al., 1992, Proc. Natl.Acad. Sci. USA 89:7958-7962). The host cell used should not express anendogenous transcription factor that binds to the same DNA site as thatrecognized by the DNA binding domain fusion population. Also,preferably, the host cell is mutant or otherwise lacking in anendogenous, functional form of the reporter gene(s) used in the assay.

Various vectors and host strains for expression of the two fusionprotein populations in yeast are known and can be used (see, e.g., U.S.Pat. No. 5,1468,614; Bartel et al., 1993, “Using the two-hybrid systemto detect protein-protein interactions” In: Cellular Interactions inDevelopment, Hartley, D. A. (ed.), Practical Approach Series xviii, IRLPress at Oxford University Press, New York, N.Y., pp. 153-179; Fieldsand Sternglanz, 1994, Trends In Genetics 10:286-292). Exemplary strainsthat can be used in the assay of the invention also include, but are notlimited to, the following:

-   -   Y190: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,        leu2-3,112, gal4α, gal80α, cyh^(r)2,        LYS2::GAL1_(UAS)-HIS3_(TATA)HIS3,        URA3::GAL1_(UAS)-GAL1_(TATA)-lacZ; Harper et al., 1993, Cell        75:805-816, available from Clontech, Palo Alto, Calif. Y190        contains HIS3 and lacZ reporter genes driven by GAL4 binding        sites.    -   CG-1945: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,        leu2-3,112, gal4-542, gal80-538, cyh^(r)2,        LYS2::GAL1_(UAS)-HIS3_(TATA)HIS3,        URA3::GAL1_(UAS17mers(x3))-CYC1_(TATA)-lacZ, available from        Clontech, Palo Alto, Calif. CG-1945 contains HIS3 and lacZ        reporter genes driven by GAL4 binding sites.    -   Y187: MAT-α, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,112,        gal4α, gal80α, URA3::GAL1_(UAS)-GAL1_(TATA)-lacZ, available from        Clontech, Palo Alto, Calif. Y187 contains a lacZ reporter gene        driven by GAL4 binding sites.    -   SFY526: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,        leu2-3,112, gal4-542, gal80-538, can^(r), URA3::GAL1-lacZ,        available from Clontech, Palo Alto, Calif. SFY526 contains HIS3        and lacZ reporter genes driven by GAL4 binding sites.    -   HF7c: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,        leu2-3,112, gal4-542, gal80-538, LYS2::GAL1-HIS3,        URA3::GAL1_(UAS 17MERS(x3))-CYC1-lacZ, available from Clontech,        Palo Alto, Calif. HF7c contains HIS3 and lacZ reporter genes        driven by GAL4 binding sites.    -   YRG-2: MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,        leu2-3,112, gal4-542, gal80-538,        LYS2::GAL1_(UAS)-GAL1_(TATA)-HIS3,        URA3::GAL1_(UAS17mers(x3))-CYC1-lacZ, available from Stratagene,        La Jolla, Calif. YRG-2 contains HIS3 and lacZ reporter genes        driven by GAL4 binding sites.

Many other strains commonly known and available in the art can be used.

If not already lacking in endogenous reporter gene activity, cellsmutant in the reporter gene may be selected by known methods, or thecells can be made mutant in the target reporter gene by knowngene-disruption methods prior to introducing the reporter gene(Rothstein, 1983, Meth. Enzymol. 101:202-211).

In a specific embodiment, plasmids encoding the different fusion proteinpopulations can be introduced simultaneously into a single host cell(e.g., a haploid yeast cell) containing one or more reporter genes, byco-transformation, to conduct the assay for protein-proteininteractions. Or, preferably, the two fusion protein populations areintroduced into a single cell either by mating (e.g., for yeast cells)or cell fusions (e.g., of mammalian cells). In a mating type assay,conjugation of haploid yeast cells of opposite mating type that havebeen transformed with a binding domain fusion expression construct(preferably a plasmid) and an activation (or inhibitor) domain fusionexpression construct (preferably a plasmid), respectively, will deliverboth constructs into the same diploid cell. The mating type of a yeaststrain may be manipulated by transformation with the HO gene (Herskowitzand Jensen, 1991, Meth. Enzymol. 194:132-146).

In a preferred embodiment, a yeast interaction mating assay is employedusing two different types of host cells, strain-type a and alpha of theyeast Saccharomyces cerevisiae. The host cell preferably contains atleast two reporter genes, each with one or more binding sites for theDNA-binding domain (e.g., of a transcriptional activator). The activatordomain and DNA binding domain are each parts of chimeric proteins formedfrom the two respective populations of proteins. One strain of hostcells, for example the a strain, contains fusions of the library ofnucleotide sequences with the DNA-binding domain of a transcriptionalactivator, such as GAL4. The hybrid proteins expressed in this set ofhost cells are capable of recognizing the DNA-binding site in thepromoter or enhancer region in the reporter gene construct. The secondset of yeast host cells, for example, the alpha strain, containsnucleotide sequences encoding fusions of a library of DNA sequencesfused to the activation domain of a transcriptional activator.

In a preferred embodiment, the fusion protein constructs are introducedinto the host cell as a set of plasmids. These plasmids are preferablycapable of autonomous replication in a host yeast cell and preferablycan also be propagated in E. coli. The plasmid contains a promoterdirecting the transcription of the DNA binding or activation domainfusion genes, and a transcriptional termination signal. The plasmid alsopreferably contains a selectable marker gene, permitting selection ofcells containing the plasmid. The plasmid can be single-copy ormulti-copy. Single-copy yeast plasmids that have the yeast centromeremay also be used to express the activation and DNA binding domainfusions (Elledge et al., 1988, Gene 70:303-312).

In another embodiment, the fusion constructs are introduced directlyinto the yeast chromosome via homologous recombination. The homologousrecombination for these purposes is mediated through yeast sequencesthat are not essential for vegetative growth of yeast, e.g., the MER2,MER1, ZIP1, REC102, or ME14 gene.

Bacteriophage vectors can also be used to express the DNA binding domainand/or activation domain fusion proteins. Libraries can generally beprepared faster and more easily from bacteriophage vectors than fromplasmid vectors.

In a specific embodiment, the present invention provides a method ofdetecting one or more protein-protein interactions comprising (a)recombinantly expressing Mam or a derivative or analog thereof in afirst population of yeast cells being of a first mating type andcomprising a first fusion protein containing the Mam sequence and a DNAbinding domain, wherein said first population of yeast cells contains afirst nucleotide sequence operably linked to a promoter driven by one ormore DNA binding sites recognized by said DNA binding domain such thatan interaction of said first fusion protein with a second fusionprotein, said second fusion protein comprising a transcriptionalactivation domain, results in increased transcription of said firstnucleotide sequence; (b) negatively selecting to eliminate those yeastcells in said first population in which said increased transcription ofsaid first nucleotide sequence occurs in the absence of said secondfusion protein; (c) recombinantly expressing in a second population ofyeast cells of a second mating type different from said first matingtype, a plurality of said second fusion proteins, each second fusionprotein comprising a sequence of a fragment, derivative or analog of aMam-IP and an activation domain of a transcriptional activator, in whichthe activation domain is the same in each said second fusion protein;(d) mating said first population of yeast cells with said secondpopulation of yeast cells to form a third population of diploid yeastcells, wherein said third population of diploid yeast cells contains asecond nucleotide sequence operably linked to a promoter driven by a DNAbinding site recognized by said DNA binding domain such that aninteraction of a first fusion protein with a second fusion proteinresults in increased transcription of said second nucleotide sequence,in which the first and second nucleotide sequences can be the same ordifferent; and (e) detecting said increased transcription of said firstand/or second nucleotide sequence, thereby detecting an interactionbetween a first fusion protein and a second fusion protein.

In a preferred embodiment, the bait Mam sequence and the prey library ofchimeric genes are combined by mating the two yeast strains on solidmedia for a period of approximately 6-8 hours. In a less preferredembodiment, the mating is performed in liquid media. The resultingdiploids contain both kinds of chimeric genes, i.e., the DNA-bindingdomain fusion and the activation domain fusion.

Preferred reporter genes include the URA3, HIS3 and/or the lacZ genes(see, e.g., Rose and Botstein, 1983, Meth. Enzymol. 101:167-180)operably linked to GAL4 DNA-binding domain recognition elements. Otherreporter genes comprise the functional coding sequences for, but notlimited to, Green Fluorescent Protein (GFP) (Cubitt et al., 1995, TrendsBiochem. Sci. 20:448-455), luciferase, LEU2, LYS2, ADE2, TRP1, CAN1,CYH2, GUS, CUP1 or chloramphenicol acetyl transferase (CAT). Expressionof LEU2, LYS2, ADE2 and TRP1 are detected by growth in a specificdefined media; GUS and CAT can be monitored by well known enzyme assays;and CAN1 and CYH2 are detected by selection in the presence ofcanavanine and cycloheximide. With respect to GFP, the naturalfluorescence of the protein is detected.

In a specific embodiment, transcription of the reporter gene is detectedby a linked replication assay. For example, as described by Vasavada etal., 1991, Proc. Natl. Acad. Sci. USA 88:10686-10690, expression of SV40large T antigen is under the control of the E1B promoter responsive toGAL4 binding sites. The replication of a plasmid containing the SV40origin of replication, indicates the reconstruction of the GAL4 proteinand a protein-protein interaction. Alternatively, a polyoma virusreplicon can be employed (Vasavada et al., 1991, Proc. Natl. Acad. Sci.USA 88:10686-10690).

In another embodiment, the expression of reporter genes that encodeproteins can be detected by immunoassay, i.e., by detecting theimmunospecific binding of an antibody to such protein, which antibodycan be labeled, or alternatively, which antibody can be incubated with alabeled binding partner to the antibody, so as to yield a detectablesignal. Alam and Cook (1990, Anal. Biochem. 188:245-254) disclosenon-limiting examples of detectable marker genes that can be operablylinked to a transcriptional regulatory region responsive to areconstituted transcriptional activator, and thus used as reportergenes.

The activation of reporter genes like URA3 or HIS3 enables the cells togrow in the absence of uracil or histidine, respectively, and henceserves as a selectable marker. Thus, after mating, the cells exhibitingprotein-protein interactions are selected by the ability to grow inmedia lacking a nutritional component, such as uracil or histidine(referred to as -URA (minus URA) and -HIS (minus HIS) medium,respectively). The -HIS medium preferably contains3-amino-1,2,4-triazole (3-AT), which is a competitive inhibitor of theHIS3 gene product, and thus, requires higher levels of transcription inthe selection (see, Durfee et al., 1993, Genes Dev. 7:555-569).Similarly, 6-azauracil, which is an inhibitor of the URA3 gene product,can be included in -URA medium (Le Douarin et al., 1995, Nucl. AcidsRes. 23:876-878). URA3 gene activity can also be detected and/ormeasured by determining the activity of its gene product,orotidine-51-monophosphate decarboxylase (Pierrat et al., 1992, Gene119:237-245, Wolcott et al., 1966, Biochem. Biophys. Acta 122:532-534).In other embodiments of the present invention, the activities of thereporter genes like GFP or lacZ are monitored by measuring a detectablesignal (e.g., fluorescent or chromogenic, respectively) that resultsfrom the activation of these reporter genes. For example, lacZtranscription can be monitored by incubation in the presence of achromogenic substrate, such as X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside), of its encoded enzyme,β-galactosidase. The pool of all interacting proteins isolated by thismanner from mating the Mam sequence product and the library identifiesthe “Mam interactive population”.

In a preferred embodiment of the present invention, false positivesarising from transcriptional activation by the DNA binding domain fusionproteins in the absence of a transcriptional activator domain fusionprotein are prevented or reduced by negative selection for suchactivation within a host cell containing the DNA binding fusionpopulation, prior to exposure to the activation domain fusionpopulation. By way of example, if such cell contains URA3 as a reportergene, negative selection is carried out by incubating the cell in thepresence of 5-fluoroorotic acid (5-FOA, which kills URA+ cells(Rothstein, 1983, Meth. Enzymol. 101:167-180). Hence, if the DNA-bindingdomain fusions by themselves activate transcription, the metabolism of5-FOA will lead to cell death and the removal of self-activatingDNA-binding domain hybrids.

Negative selection involving the use of a selectable marker as areporter gene and the presence in the cell medium of an agent toxic orgrowth inhibitory to the host cells in the absence of reporter genetranscription is preferred, since it allows a higher rate of processingthan other methods. As will be apparent, negative selection can also becarried out on the activation domain fusion population prior tointeraction with the DNA binding domain fusion population, by similarmethods, either alone or in addition to negative selection of the DNAbinding fusion population.

Negative selection can also be carried out on the recovered Mam:Mam-IPcomplex by known methods (see, e.g., Bartel et al., 1993, BioTechniques14:920-924) although pre-negative selection (prior to the interactionassay), as described above, is preferred. For example, each plasmidencoding a protein (peptide or polypeptide) fused to the activationdomain (one-half of a detected interacting complex) can be transformedback into the original screening strain, either alone or with a plasmidencoding only the DNA-binding domain, the DNA-binding domain fused tothe detected interacting protein, or the DNA-binding domain fused to aprotein that does not affect transcription or participate in theprotein-protein interaction. A positive interaction detected with anyplasmid other than that encoding the DNA-binding domain fusion to thedetected interacting protein is deemed a false positive and iseliminated from the screen.

In a preferred embodiment, the Mam plasmid population is transformed ina yeast strain of a first mating type (a or alpha), and the secondplasmid population (containing the library of DNA sequences) istransformed in a yeast strain of a different mating type. Both strainsare preferably mutant for URA3 and HIS3, and contain HIS3, andoptionally lacZ, as reporter genes. The first set of yeast cells arepositively selected for the Mam plasmids and are negatively selected forfalse positives by incubation in medium lacking the selectable marker(e.g., tryptophan) and containing 5-FOA. Yeast cells of the secondmating type are transformed with the second plasmid population, and arepositively selected for the presence of the plasmids containing thelibrary of fusion proteins. Selected cells are pooled. Both groups ofpooled cells are mixed together and mating is allowed to occur on asolid phase. The resulting diploid cells are then transferred toselective media that selects for the presence of each plasmid and foractivation of reporter genes.

In a preferred embodiment of the invention, after an interactivepopulation is obtained, the DNA sequences encoding the pairs ofinteractive proteins are isolated by a method wherein either theDNA-binding domain hybrids or the activation domain hybrids areamplified, in separate respective reactions. Preferably, theamplification is carried out by polymerase chain reaction (PCR) (see,U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,889,818; Gyllenstein et al.,1988, Proc. Natl. Acad. Sci. USA 85:7652-7656; Ochman et al., 1988,Genetics 120:621-623; Loh et al., 1989, Science 243:217-220; Innis etal., 1990, PCR Protocols, Academic Press, Inc., San Diego, Calif.) usingpairs of oligonucleotide primers specific for either the DNA-bindingdomain hybrids or the activation domain hybrids. This PCR reaction canalso be performed on pooled cells expressing interacting proteincomplexes, preferably pooled arrays of interactants. Other amplificationmethods known in the art can be used, including but not limited toligase chain reaction (see EP 320,308), use of Qβ replicase, or methodslisted in Kricka et al., 1995, Molecular Probing, Blotting, andSequencing, Academic Press, New York, Chapter 1 and Table IX.

The plasmids encoding the DNA-binding domain hybrid and the activationdomain hybrid proteins can also be isolated and cloned by any of themethods well known in the art. For example, but not by way oflimitation, if a shuttle (yeast to E. coli) vector is used to expressthe fusion proteins, the genes can be recovered by transforming theyeast DNA into E. coli and recovering the plasmids from E. coli (see,e.g., Hoffman et al., 1987, Gene 57:267-272). Alternatively, the yeastvector can be isolated, and the insert encoding the fusion proteinsubcloned into a bacterial expression vector, for growth of the plasmidin E. coli.

5.9 Pharmaceutical Compositions and Therapeutic/ProphylacticAdministration

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal including, but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a Mammal, and most preferably human. In a specificembodiment, a non-human Mammal is the subject.

Formulations and methods of administration that can be employed when theTherapeutic comprises a nucleic acid are described in Sections 5.5.2 and5.5.3, supra; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, and microcapsules: use of recombinant cells capable ofexpressing the Therapeutic, use of receptor-mediated endocytosis (e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432); construction of aTherapeutic nucleic acid as part of a retroviral or other vector, etc.Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion, by bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oral,rectal and intestinal mucosa, etc.), and may be administered togetherwith other biologically active agents. Administration can be systemic orlocal. In addition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment. This may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome (Langer, 1990, Science 249:1527-1533; Treat etal., 1989, In: Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler, eds., Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the Therapeutic can be delivered via acontrolled release system. In one embodiment, a pump may be used(Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201-240;Buchwald et al., 1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl.J. Med. 321:574-579). In another embodiment, polymeric materials can beused (Medical Applications of Controlled Release, Langer and Wise, eds.,CRC Press, Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball, eds., Wiley, New York,1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190-192; During et al., 1989, Ann.Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863). Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (e.g., Goodson, 1984, In: MedicalApplications of Controlled Release, supra, Vol. 2, pp. 115-138). Othercontrolled release systems are discussed in the review by Langer (1990,Science 249:1527-1533).

In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein Therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or bycoating it with lipids, cell-surface receptors or transfecting agents,or by administering it in linkage to a homeobox-like peptide which isknown to enter the nucleus (e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeuticcan be introduced intracellularly and incorporated by homologousrecombination within host cell DNA for expression.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly, in humans. The term “carrier” refers toa diluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, including but not limited to peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered orally. Saline andaqueous dextrose are preferred carriers when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions are preferably employed as liquidcarriers for injectable solutions. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theTherapeutic, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated, in accordancewith routine procedures, as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water or saline forinjection can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freecarboxyl groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., those formed with free aminegroups such as those derived from isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc., and those derived fromsodium, potassium, ammonium, calcium, and ferric hydroxides, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.10 Animal Models

The present invention also provides animal models. In one embodiment,animal models for diseases and disorders involving Mam:Mip1, Mam:Mip30or Mam:Mip6 complexes are provided. These include, but are not limitedto, disease or disorders of cell fate and differentiation and disordersassociated with aberrant mitosis, see Section 2, supra. Such animals canbe initially produced by promoting homologous recombination orinsertional mutagenesis between Mam, Mip1, Mip30 and/or Mip6 genes inthe chromosome, and exogenous Mam, Mip1, Mip30 and/or Mip6 genes thathave been rendered biologically inactive or deleted (preferably byinsertion of a heterologous sequence, e.g., an antibiotic resistancegene). In a preferred aspect, homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with a vector containing theinsertionally inactivated Mam, Mip1, Mip30 and/or Mip6 genes, such thathomologous recombination occurs, followed by injecting the transformedES cells into a blastocyst, and implanting the blastocyst into a fostermother, followed by the birth of the chimeric animal (“knockout animal”)in which a Mam, Mip1, Mip30 and/or Mip6 gene has been inactivated ordeleted (Capecchi, 1989, Science 244:1288-1292). In another preferredaspect, site-specific recombinases can be used, such as cre whichrecognizes lox sites and flp which recognizes frt sites. The chimericanimal can be bred to produce additional knockout animals. Such animalscan be mice, hamsters, sheep, pigs, cattle, etc., and are preferablynon-human Mammals. In a specific embodiment, a knockout mouse isproduced.

Such knockout animals are expected to develop, or be predisposed todeveloping, diseases or disorders involving, but not restricted to,diseases and disorder of cell fate and differentiation, and a number ofless common syndromes and disorders associated with aberrant mitoticevents, and thus, can have use as animal models of such diseases anddisorders, e.g., to screen for or test molecules (e.g., potentialTherapeutics) for diseases or disorders of cell fate anddiffernentiation, e.g., hyperproliferative disorders and malignacies.

In a different embodiment of the invention, transgenic animals that haveincorporated and express (or overexpress or mis-express) a functionalMam, Mip1, Mip30 and/or Mip6 gene, e.g. by introducing the Mam and Mip1genes under the control of a heterologous promoter (i.e., a promoterthat is not the native Mam or Mip1 promoter) that either overexpressesthe protein or proteins, or expresses them in tissues not normallyexpressing the complexes or proteins, can have use as animal models ofdiseases and disorders characterized by elevated levels of Mam:Mip1complexes. Such animals can be used to screen or test molecules for theability to treat or prevent the diseases and disorders cited supra.

In one embodiment, the present invention provides a recombinantnon-human animal in which both an endogenous Mam gene and an endogenousMip1 have been deleted or inactivated by homologous recombination orinsertional mutagenesis of said animal or an ancestor thereof. Inanother embodiment, the invention provides a recombinant non-humananimal containing both a Mam gene and a Mip1 gene in which the Mam geneis under the control of a promoter that is not the native Mam genepromoter and the Mip1 gene is under the control of a promoter that isnot the native Mip 1 gene promoter. In a specific embodiment, theinvention provides a recombinant non-human animal containing a transgenecomprising a nucleic acid sequence encoding a chimeric proteincomprising a fragment of Mam of at least 6 amino acids fused via acovalent bond to a fragment of Mip1 protein of at least 6 amino acids.

6. EXAMPLES

6.1 Identification of Mam Interactions

To elucidate the function of Mam and its role in Notch signaling,proteins with which Mam interacts were identified. Complementary DNAencoding a truncated Mam protein, in which the carboxy-terminal 32 aminoacids of full-length Mam were deleted, was fused to the Gal4 DNA-bindingdomain encoded by the yeast expression vector pEG202. A truncated Mamfusion protein was used because it elicited a lower autonomoustransactivational response from yeast reporter genes than full-lengthMam. Using this fusion protein as bait, approximately 3×10⁶ yeasttransformants expressing proteins encoded by Drosophila cDNAs werescreened, prepared from 0-12 hour embryos, fused to the E. coli B42transactivation domain encoded by pJG4-5. FIG. 13 is a graph showing theresults of a yeast two-hybrid screen deomonstrating that Mam interactswith Mip1, Mip30 and Mip6.

Three cDNAs encoding Mam-Interacting Proteins (Mips) were isolated. Thelargest cDNA encoding one of the interacting proteins, Mip1, was 862nucleotides in length, included 19 poly (A) residues at its 3′ end, andpredicted an amino-terminally truncated protein of 242 amino acids. Toobtain additional 5′ sequence, the Mip1 cDNA isolated from thetwo-hybrid library was used as a probe to screen a lambda phage cDNAlibrary prepared from 0-14 hour embryos. The largest Mip1 cDNA cloneisolated from this library was 2072 nucleotides in length, waspolyadenylated at an identical position, and encoded a protein of 411amino acids that also appeared to be truncated at its amino terminus.The remaining amino-terminal sequence of the Mip1 protein was identifiedin the sequences of EST and baculoviris clones, deposited in GenBank,and was isolated by PCR from a lambda phage library. The entire Mip1cDNA is 2348 nucleotides in length, and the largest open reading frameencodes a protein of 700 amino acids with a predicted molecular mass of78 kD. The size of this cDNA agrees closely with the size of the singletranscript detected by northern blot analysis.

6.2 Characterization of Mip1, Mip30 and Mip6

A search of the Mip1 protein sequence for profiles or patterns using theInterPro Scan program identified three prominent signature motifs. Theseare an UBA/THIF-type NAD/FAD binding fold (THIF family), anubiquitin-activating enzyme repeat domain (UBACT repeat), and abipartite nuclear localization signal (FIG. 5). Each of these motifs isevolutionarily conserved and found in organisms ranging from human tobacteria (THIF family) or yeast (UBACT family). In eukaryotes, thesemotifs are present in ubiquitin-activating enzymes (E1-type enzymes).E1-type enzymes activate ubiquitin or ubiquitin-related proteins, firstby adenylating a C-terminal glycine residue with AT2, and then byforming a thicester linkage between the ubiquitin or ubiquitin-relatedprotein and a cysteine residue of the E1 enzyme, releasing AMP. Theubiquitin or ubiquitin-related moiety is subsequently seriallytransferred to a cysteine residue of an ubiquitin-conjugating enzyme (E2enzyme), a cysteine residue of a ubiquitin ligase, and then ultimately alysine residue of a target protein.

Using the BLAST program to search GenBank for sequence similaritiesrevealed that Mip1 is the Drosophila ubiquitin-like activating enzymeUba2p. This protein is one subunit of a heterodixneric E1-type enzymethat activates the Small Ubiquitin-related Modifier, SUMO/Smt3, and isextremely well conserved throughout evolution. An alignment of theDrosophila and human proteins using the CUSTALW program indicates thatthey are 46% identical (FIG. 7).

The SUMO-conjugation machinery appears to be present in all Eukaryotaand mechanistically parallels the ubiquitin-conjugation machinery. Thenotable differences are that the E1 enzyme of the ubiquitin-conjugationpathway is composed of a single protein, while the E1 enzyme of theSUMO-conjugation pathway is composed of two subunits: Aos1p (anotherTHIF-family protein) and Uba2p (Mip1). These two proteins are equivalentto the amino- and carboxy-terminal regions of the classic ubiquitin E1enzyme, respectively. Additionally, an E3-type protein ligase has notyet been identified as a component of the SUMO-conjugation machinery.

The cDNA encoding Mip30, isolated from the two-hybrid screen, was 1818nucleotides in length, included 18 poly(A) residues at its 3′ end, andpredicted an amino-terminally truncated protein of 315 amino acids. Toobtain additional 5′ sequence, the Mip30 cDNA isolated from thetwo-hybrid library was used as a probe to screen a lambda phage cDNAlibrary. An additional 2560 nucleotides of 5′ sequence was obtained. Theremaining 5′ sequence (nucleotides 1-7) of Mip30 was identified in thesequence of EST clones (AA948812, AA940874 and AA949252) deposited inGenBank. The entire Mip30 sequence is 2567 nucleotides in length, andthe largest open reading frame encodes a protein of 543 amino acids witha predicted molecular mass of 63 kD, corresponding to the proteinpredicted by AF132187. Sequence analyses of lambda phage clones revealedthree forms of Mip30 cDNA that differ in the length of their3′-untranslated regions. The predicted size of the transcripts agreesclosely with the sizes predicted by Northern blot analysis. A search ofthe Mip30 protein sequence for profiles or patterns using the InterProScan program identified three prominent signature motifs. These areseven C2H2-type zinc fingers, an HMG-1 and HMG-Y DNA-binding domain(A+T-hook), and a bipartite nuclear localization signal. See FIG. 11.

The cDNA encoding Mip6, isolated from the two-hybrid screen, was 1224nucleotides in length, included 20 poly(A) residues at its 3′ end, andpredicted an amino-terminally truncated protein of 251 amino acids. Toobtain additional 5′ sequence, the Mip6 cDNA isolated from thetwo-hybrid library was used as a probe to screen a lambda phage cDNAlibrary. An additional 35 nucleotides (11 amino acids) of 5′ sequencewas obtained from this clone. The remaining amino-terminal sequence ofthe Mip1 protein was identified in the sequence of EST (BF503916) andbaculovirus (AC008326 and AC007977) clones, as well as the Drosophilagenomic scaffold (AE003615), deposited in GenBank. The entire Mip6sequence is 2140 nucleotides in length, and the largest open readingframe encodes a protein of 625 amino acids with a predicted molecularmass of 69 kD, corresponding to the conceptual translation AAF52468. Thesize of this cDNA agrees closely with the size of the single transcriptdetected by northern blot analysis. The only identifiable motif in theMip6 protein is a bipartite nuclear localization signal (amino acids420-437). See FIG. 12.

6.3 Subcellular Localization of Mastermind

Indirect immunofluorescence analysis of human 293T cells, Drosophila S2cells or Spodoptera SF9 cells transiently transfected with DrosophilaMastermind (Mam), epitope-tagged at its amino terminus with either Flagor hemagglutinin (HA) revealed that Mam localizes to discrete subnucleardomains (FIG. 14). A similar subnuclear localization has been found fora human protein, MAML1, that shares limited homology with DrosophilaMam. Co-localization in 293T cells with antibodies to the promyelocyticleukemia (PML.) protein identified these domains as nuclear bodies (NBs)(FIG. 15). Nuclear bodies (also called PML bodies, PODs or ND10) aregeneral features of cells; there is typically 5-20 per cell, they rangein size from 0.1-1μ, and they are spherical or toroidal in shape. Manytypes of proteins have been found to co-localize with NBs, includingtranscription factors and coactivators, chromosomal proteins, tumorsuppressors and proto-oncogenes. Interestingly, the SUMO protein hasbeen shown to be associated with NBs, and components of NBs, such as thesignature proteins PML and SP100, are conjugated to SUMO, see Section 2,supra. A relationship between NBs and disease is exemplified by theirdisruption in malignancies, such as acute promyelocytic leukemia, andupon viral infection. The function of NBs is still unknown, but basedupon the variety of proteins that are found associated with NBs, twofavored hypotheses are that these structures are sites for signalintegration or sites for protein storage/removal.

The localization of Mam to NBs indicated that Mam recruits othercomponents of the Notch pathway to these structures. Co-expression ofepitope-tagged Mam and intracellular Notch in 293T cells demonstratedthat Mam causes the relocalization of intracellular Notch from thenucleoplasm to NBs (FIG. 16). A similar observation has been reportedfor the human MAML1 protein, and studies have shown that mammalianMam-like proteins as well as the C. elegans LAG-3 protein, apolyglutamine rich protein, interact with the ankyrin repeats of Notch(Wu et al., 2000, Nature Genetics 26:484-489). Furthermore, theseproteins form a ternary complex with the intracellular domain of Notchand the downstream effector Su(H)/RBP-j/LAG1, and function as activatorsof Notch signal transduction. Taken together, these observationsindicate a functional relationship between NBs and Notch signaling.

6.4 Mastermind Recruits Mip1 to NBs

Co-expression of an epitope-tagged Mam protein with an epitope-taggedMip1 protein, in transiently transfected 293T cells, demonstrated thatMam recruits Mip1 to NBs (FIG. 17). When Mip1 is expressed alone, itsdistribution appears to be homogenous throughout the nucleoplasm;however, when co-expressed with Mam, Mip1 can be seen in discretesubnuclear domains. This observation confirms the interaction betweenMam and Mip1 observed in the two-hybrid screen and suggests that Mamand, consequently, Notch signaling integrates into the SUMO-conjugationpathway.

6.5 Mam Activates SUMO Conjugation

Two possible scenarios can be envisioned to explain the interactionbetween Mam and a component of the SUMO-conjugation pathway. Onescenario would have Mam as a target of the SUMO conjugation machinery,but we have not yet identified a SUMO-conjugated form of Mam. This couldbe explained by the observations that certain SUMO conjugates appear tobe very unstable and that only a small percentage of certain targetproteins exist in a conjugated form. However, most proteins that areconjugated to SUMO are found to directly interact with the SUMOconjugating enzyme (Ubc9) or the SUMO protein itself, and we have notobserved such interactions. A second scenario would have Mam influencethe activity of the SUMO-conjugation machinery. Given that Mam interactswith the most upstream enzyme of the SUMO-conjugating apparatus andinfluences the intracellular distribution of one of its subunits, wesought to test this possibility.

To investigate a potential role of Mam in SUMO conjugation, we exploitedthe extremely high evolutionary conservation of the SUMO conjugationmachinery and the high transfectability of 293T cells. Complementary DNAencoding Drosophila SUMO was isolated from a lambda phage library byPCR, fused at its amino terminus to sequence encoding the HA epitope andcloned into an expression vector. When expressed in 293T cells,Drosophila SUMO was efficiently conjugated to cellular proteins, but ata low level. However, when Drosophila SUMO was co-expressed withDrosophila Mam, a dramatic increase in the extent of SUMO conjugation tocellular targets was observed. Furthermore, this increase was directlyproportional to the amount of Mam-encoding plasmid that was introducedinto these cells (FIG. 18).

As a corollary to the above experiment, we cloned cDNA encodingDrosophila SUMO and Mam into a baculovirus production vector, pFastBac,to efficiently express these protein in insect cells. Again, we foundthat Drosophila SUMO was efficiently conjugated to SF9 cellular proteinsand the extent of conjugation was proportional to the amount of Mamexpressed.

These results demonstrate that Mam can positively regulate the SUMOconjugation machinery. Taken in light of the interaction data, theseobservations also indicate that the activity of Mam resides at thehighest level of the SUMO-conjugating apparatus.

6.6 Mam is a General Activator of the SUMO Conjugation Machinery

The genome of Drosophila predicts only one SUMO-encoding gene. However,at least three SUMO encoding genes have been identified in the genomesof mammals (SUMO1/SUMOC, SUMO2/SUMOA, and SUMO3/SUMOB). Nucleic andamino acid sequences for the human homologs of SUMOA, SUMOB, and SUMOCare found in GenBank under Accession Nos. X99584, X99585, and X99586,respectively. Drosophila SUMO is more closely related to mammalianSUMO2. We, therefore, wished to determine whether the activity of Mamupon the SUMO conjugating machinery was specific to one SUMO class orwas general. Accordingly, PCR was used to isolate, clone, and epitopetag cDNA encoding each of the mammalian SUMO proteins. Co-expression ofthese cDNAs with Drosophila Mam revealed that Mam increases theconjugation of each of the mammalian SUMO proteins to cellular targets(FIG. 19). Therefore, Mam appears to impinge upon the SUMO conjugationmachinery in a general manner.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of the present invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

1. A method of inhibiting Notch signal transduction in a cell comprisingcontacting the cell with an antagonist of sumolation in an amountsufficient to inhibit Notch signal transduction.
 2. A method ofagonizing Notch signal transduction in a cell comprising contacting thecell with an agonist of sumolation in an amount sufficient to agonizeNotch signal transduction.
 3. The method according to claim 1 in whichthe antagonist is a dominant negative form of Mip1.
 4. The methodaccording to claim 3 in which the dominant negative form of Mip1contains a mutated ADP binding site such that the dominant negative formof Mip1 does not bind ADP.
 5. The method according to claim 1 in whichthe antagonist is an antisense nucleic acid to Mip1, or an antibody toMip1 or the binding domain of an antibody to Mip1.
 6. A method ofidentifying a molecule that alters Notch signal transduction in a cellcomprising the following steps in the order stated: (a) contacting thecell with one or more candidate molecules; and (b) measuring the amountof sumolation in the cell, wherein an increase or decrease in the amountof sumolation relative to said amount in a cell not so contacted withone or more of the candidate molecules indicates that the candidatemolecules alter Notch signal transduction.
 7. (canceled)
 8. (canceled)9. A method of identifying a molecule that alters sumolation activity ina cell comprising the following steps in the order stated: (a)contacting the cell with one or more candidate molecules; and (b)measuring the amount of Notch signal transduction in the cell, whereinan increase or decrease in the amount of Notch signal transductionrelative to said amount in a cell not so contacted with one or more ofthe candidate molecules indicates that the candidate molecules altersumolation activity.
 10. (canceled)
 11. (canceled)
 12. A method ofinhibiting sumolation activity in a cell comprising contacting the cellwith an antagonist of Notch signal transduction in an amount sufficientto inhibit sumolation activity.
 13. A method of agonizing sumolationactivity in a cell comprising contacting the cell with an agonist ofNotch signal transduction in an amount sufficient to agonize sumolationactivity.
 14. The method according to claim 12 in which the antagonistis a dominant negative form of Notch.
 15. The method according to claim12 in which the antagonist is an antibody to Notch or a fragment of theantibody containing the binding domain of the antibody.
 16. The methodaccording to claim 13 in which the agonist is an dominant active form ofNotch.
 17. The method according to claim 13 in which the agonist is aDelta or Serrate protein or a fragment of Delta or Serrate that binds toNotch.
 18. The method according to claim 13 in which the agonist is thesoluble extracellular domain of Delta.
 19. (canceled)
 20. A purifiedcomplex of Mam and Mip1, or a purified complex of Mam and Mip30, or apurified complex of Mam and Mip6.
 21. (canceled)
 22. A purified complexselected from the group consisting of a complex of a derivative of Mamand Mip1, a complex of Mam and a derivative of Mip1, and a complex of aderivative of Mam and a derivative of Mip1; in which the derivative ofMam is able to form a complex with a wild-type Mip1 and the derivativeof Mip1 is able to form a complex with wild-type Mam.
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. A chimeric protein comprising a fragmentof Mam consisting of at least 6 amino acids fused via a covalent bond toa fragment of Mip1 consisting of at least 6 amino acids.
 27. (canceled)28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled) 32.(canceled)
 33. (canceled)
 34. (canceled)
 35. An antibody whichimmunospecifically binds the complex according to claim 20 or a fragmentor derivative of said antibody containing the binding domain thereof.36. The antibody according to claim 35 which does not immunospecificallybind Mam, Mip1, Mip30 or Mip6 that is not part of a Mam:Mip1, Mam:Mip30or Mam:Mip6 complex, respectively.
 37. An isolated nucleic acid or anisolated combination of nucleic acids comprising (a) a nucleotidesequence encoding Mam and a nucleotide sequence encoding Mip1, (b) anucleotide sequence encoding Mam and a nucleotide sequence encodingMip30, (c) a nucleotide sequence encoding Mam and a nucleotide sequenceencoding Mip6, (d) a nucleotide sequence encoding Mip30, or (e) anucleotide sequence encoding Mip6.
 38. (canceled)
 39. (canceled) 40.(canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled)
 44. (canceled)45. (canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled) 49.(canceled)
 50. (canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled)54. (canceled)
 55. (canceled)
 56. A method of diagnosing or screeningfor the presence of or a predisposition for developing a disease ordisorder characterized by an aberrant level of a complex of Mam andMip1, Mam and Mip30 or Mam and Mip6, in a subject comprising measuringthe level of said complex, RNA encoding Mam and Mip1, Mam and Mip30 orMam and Mip6, or functional activity of said complex in a sample derivedfrom the subject, in which an increase or decrease in the level of saidcomplex, said RNA encoding Mam and Mip1, Mam and Mip30 or Mam and Mip6,or functional activity of said complex in the sample, relative to thelevel of said complex, said RNA encoding Mam and Mip1, Mam and Mip30 orMam and Mip6 or functional activity of said complex found in ananalogous sample not having the disease or disorder or a predispositionfor developing the disease or disorder, indicates the presence of thedisease or disorder or a predisposition for developing the disease ordisorder.
 57. (canceled)
 58. (canceled)
 59. (canceled)
 60. (canceled)61. (canceled)
 62. (canceled)
 63. (canceled)
 64. (canceled)
 65. A methodof treating or preventing a disease or disorder involving an aberrantlevel of Mip1, Mip30 or Mip6 in a subject comprising administering to asubject in which such treatment or prevention is desired atherapeutically effective amount of a molecule that modulates thefunction of Mip1, Mip30 or Mip6, respectively.
 66. (canceled) 67.(canceled)
 68. (canceled)
 69. (canceled)
 70. (canceled)
 71. (canceled)72. A method of treating or preventing a disease or disorder involvingan aberrant level of Mam in a subject comprising administering to asubject in which such treatment or prevention is desired atherapeutically effective amount of a molecule that modulates thefunction of Mam.
 73. (canceled)
 74. (canceled)
 75. (canceled) 76.(canceled)
 77. (canceled)
 78. (canceled)
 79. A recombinant non-humananimal in which both an endogenous Mam gene and an endogenous Mip1 havebeen deleted or inactivated by recombination or insertional mutagenesisof said animal or an ancestor thereof.
 80. (canceled)
 81. (canceled) 82.(canceled)
 83. (canceled)
 84. (canceled)
 85. (canceled)
 86. (canceled)87. (canceled)
 88. (canceled)
 89. (canceled)
 90. (canceled) 91.(canceled)
 92. (canceled)
 93. (canceled)
 94. (canceled)
 95. (canceled)96. (canceled)
 97. (canceled)
 98. (canceled)
 99. (canceled) 100.(canceled)
 101. (canceled)
 102. (canceled)
 103. (canceled) 104.(canceled)
 105. (canceled)
 106. (canceled)
 107. (canceled)
 108. A methodof monitoring the efficacy of a treatment of a disease or disordercharacterized by an aberrant level of a complex of Mam and Mip1 in asubject administered said treatment for said disease or disordercomprising measuring the level of said complex, RNA encoding Mam andMip1, or functional activity of said complex in a sample derived fromsaid subject wherein said sample is taken from said subject after theadministration of said treatment and compared to (a) said level in asample taken from said subject prior to the administration of thetreatment or (b) a standard level associated with the pretreatment stageof the disease or disorder, in which the change, or lack of change inthe level of said complex, said RNA encoding Mam and Mip1, or functionalactivity of said complex in said sample taken after the administrationof said treatment relative to the level of said complex, said RNAencoding Mam and Mip1 or functional activity of said complex in saidsample taken before the administration of said treatment or to saidstandard level indicates whether said administration is effective fortreating said disease or disorder.
 109. (canceled)
 110. (canceled) 111.A purified protein selected from the group consisting of Mip30 and Mip6.112. (canceled)
 113. (canceled)
 114. (canceled)
 115. (canceled)
 116. Apurified fragment of a Mip30 protein comprising a domain of the proteinselected from the group consisting of the C2H2-type zinc finger domain,the HMG-1 and HMG-Y DNA-binding domain (A+T-hook), and the bipartitenuclear localization signal.
 117. (canceled)
 118. (canceled) 119.(canceled)
 120. (canceled)
 121. A chimeric protein comprising a fragmentof a Mip6 protein consisting of at least 20 amino acids fused via acovalent bond to an amino acid sequence of a second protein, in whichthe second protein is not the Mip6 protein.
 122. (canceled) 123.(canceled)
 124. (canceled)
 125. (canceled)
 126. An antibody which iscapable of binding the Mip30 protein of claim
 111. 127. An antibodywhich is capable of binding the Mip6 protein of claim
 111. 128.(canceled)
 129. (canceled)
 130. (canceled)
 131. (canceled) 132.(canceled)
 133. (canceled)
 134. (canceled)
 135. (canceled) 136.(canceled)
 137. (canceled)
 138. (canceled)
 139. (canceled)
 140. A methodof treating or preventing a disease or disorder in a subject comprisingadministering to a subject in which such treatment or prevention isdesired a therapeutically effective amount of a Mip30 or Mip6 protein orderivative thereof which is able to bind to a Mam protein. 141.(canceled)
 142. (canceled)
 143. A method of treating or preventing adisease or disorder in a subject comprising administering to a subjectin which such treatment or prevention is desired a therapeuticallyeffective amount of a molecule, in which the molecule is anoligonucleotide which (a) consists of at least six nucleotides; (b)comprises a sequence complementary to at least a portion of an RNAtranscript of a Mip30 or a Mip6 gene; and (c) is specificallyhybridizable to the RNA transcript.
 144. (canceled)
 145. An isolatedoligonucleotide consisting of at least six nucleotides, and comprising asequence complementary to at least a portion of an RNA transcript of aMip30 or Mip6 gene, which oligonucleotide is specifically hybridizableto the RNA transcript.
 146. (canceled)
 147. (canceled)